Human c-Maf compositions and methods of use thereof

ABSTRACT

Isolated nucleic acid molecules encoding human c-Maf, and isolated c-Maf proteins, are provided. The invention further provides antisense nucleic acid molecules. recombinant expression vectors containing a nucleic acid molecule of the invention, host cells into which the expression vectors have been introduced and non-human transgenic animals carrying a human c-Maf transgene. The invention further provides human c-Maf fusion proteins and anti-human c-Maf antibodies. Methods of using the human c-maf compositions of the invention are also disclosed, including methods for detecting human c-Maf activity in a biological sample, methods of modulating human c-Maf activity in a cell, and methods for identifying agents that modulate the activity of human c-Maf.

RELATED APPLICATIONS

This application is a divisional of Ser. No. 09/086,010, filed May 27,1998, now U.S. Pat. No. 6,274,338, which a continuation in part of Ser.No. 09/030,579, filed Feb. 24, 1998, now abandoned.

GOVERNMENT FUNDING

Work described herein was supported, at least in part, under grant AI/AG37833 awarded by the National Institutes of Health. The U.S. governmenttherefore may have certain rights in this invention.

BACKGROUND OF THE INVENTION

The Maf family of proteins are a sub-family of AP-1/CREB/ATF proteins.The first member of the family to be identified, the v-maf oncogene, wasoriginally isolated from a spontaneous musculoaponeurotic fibrosarcomaof chicken and identified as the transforming gene of the avianretrovirus, AS42 (Nishizawa, M. et al. (1989) Proc. Natl. Acad. Sci. USA86:7711-7715). V-maf encodes a 42 kd basic region/leucine zipper (b-zip)protein with homology to the c-fos and c-jun oncogenes. Its cellularhomologue, the c-maf proto-oncogene, which has been isolated from murinecells, has only two structural changes in the coding region from v-maf(Kataoka, K. et al. (1993) J. Virol. 67:2133-2141). The maf familyincludes c-Maf, mafB, a human retina-specific protein Nrl (Swaroop, A.et al. (1992) Proc. Natl. Acad. Sci. USA 89:266-270), mafK, mafF, mafGand p18. The latter four, mafK, mafF, mafG and p18, each encode proteinsthat lack the amino terminal two thirds of c-Maf that contains thetransactivating domain (“small maf proteins”) (Fujiwara, K. T. et al.(1993) Oncogene 8:2371-2380; Igarashi, K. et al. (1995) J. Biol. Chem.270:7615-7624; Andrews, N. C. et al. (1993) Proc. Natl. Acad. Sci. USA90:11488-11492; Kataoka, K. et al. (1995) Mol. Cell. Biol.15:2180-2190).

C-Maf and other Maf family members form homodimers and heterodimers witheach other and with Fos and Jun, consistent with the known ability ofthe AP-1 proteins to pair with each other (Kerppola, T. K. and Curran,T. (1994) Oncogene 9:675-684; Kataoka, K. et al. (1994) Mol. Cell. Biol.14:700-712). The DNA target sequence to which c-Maf homodimers bind,termed the c-Maf response element (MARE), is a 13 or 14 bp element whichcontains a core TRE (T-MARE) or CRE (C-MARE) palindrome respectively.c-Maf has been shown to stimulate transcription from the Purkinjeneuron-specific promoter L7 (Kurscher, C. and Morgan, J. I. (1994) Mol.Cell. Biol. 15:246-254) and Nrl has been shown to drive expression ofthe QR1 retina-specific gene (Swaroop, A. et al. (1992) Proc. Natl.Acad. Sci. USA 89:266-270). Additionally, the small mafs have been shownto function as repressors of α and β-globin transcription when bound ashomodimers but are essential as heterodimeric partners with theerythroid-specific factor p45NF-E2 to activate globin gene transcription(Kataoka, K. et al (1995) Mol. Cell. Biol. 15:2180-2190; Igarashi, K. etal. (1994) Nature 367:568-572). MafK overexpression has been shown toinduce erythroleukemia cell differentiation (Igarashi, K. et al. (1995)Proc. Natl. Acad. Sci. USA 92:7445-7449). Moreover, c-Maf has been shownto control the tissue-specific expression of the cytokine interleukin-4in T helper 2 (Th2) cells (Ho, I-C. et al. (1996) Cell 85:973-983).

The nucleotide sequence of the mouse c-maf proto-oncogene, and predictedamino acid sequence for the mouse c-Maf protein, have been described(Kurscher, C. and Morgan, J. I. (1995) Mol. Cell. Biol. 15:246-254; andGenbank Accession number S74567). The nucleotide sequence of the chickenc-maf proto-oncogene, and predicted amino acid sequence for the chickenc-Maf protein, also have been described (Kataoka et al., GenbankAccession number D28596). However, these non-human c-Maf compositionsmay not function optimally in human cells and, moreover, use of thesecompositions in humans is likely to stimulate an immune response, sincethe chicken or mouse c-Maf would be recognized as “foreign” by the humanimmune system. Accordingly, there is still a need for human c-Mafcompositions that are suitable for use in humans.

SUMMARY OF THE INVENTION

This invention provides human c-Maf compositions. In particular, thisinvention provides isolated nucleic acid molecules encoding human c-Mafand isolated human c-Maf protein. Since the c-Maf compositions of theinvention are human-derived, they function optimally in human cells(compared with non-human c-Maf compositions) and do not stimulate animmune response in humans.

One aspect of the invention pertains to an isolated nucleic acidmolecule comprising a nucleotide sequence encoding human c-Maf. In apreferred embodiment, the nucleic acid molecule comprises the nucleotidesequence of the coding region of the NheI/XbaI insert of plasmidpHu-c-Maf (ATCC Accession No. 98671). In another preferred embodiment,the nucleic acid molecule comprises the nucleotide sequence of SEQ IDNO: 1. In other embodiments, the nucleic acid molecule has at least 98%nucleotide identity, more preferably 99% nucleotide identity, and evenmore preferably 99.5% nucleotide identity with the nucleotide sequenceof SEQ ID NO: 1 or the nucleotide sequence of the NheI/XbaI insert ofplasmid pHu-c-Maf (ATCC Accession No. 98671). In yet another embodiment,the nucleic acid molecule comprises the nucleotide sequence of theNheI/XbaI insert of plasmid pHu-c-Maf (ATCC Accession No. 98671).

The isolated nucleic acid molecules of the invention encoding humanc-Maf can be incorporated into a vector, such as an expression vector,and this vector can be introduced into a host cell. The invention alsoprovides a method for producing a human c-Maf protein by culturing ahost cell of the invention (carrying a hu-c-Maf expression vector) in asuitable medium until a human c-Maf protein is produced. The method canfurther involve isolating the human c-Maf protein from the medium or thehost cell.

Another aspect of the invention pertains to an isolated human c-Mafprotein. Preferably, the human c-Maf protein comprises the amino acidsequence encoded by the coding region of the NheI/XbaI insert of plasmidpHu-c-Maf (ATCC Accession No. 98671). In another preferred embodiment,the protein comprises the amino acid sequence of SEQ ID NO: 2. In otherembodiments, the protein has at least 98% amino acid identity, morepreferably 99% amino identity, and even more preferably 99.5% amino acididentity with SEQ ID NO: 2 or the protein encoded by the coding regionof the NheI/XbaI insert of plasmid pHu-c-Maf (ATCC Accession No. 98671).

Fusion proteins, comprising a human c-Maf protein operatively linked toa polypeptide other than human c-Maf, are also encompassed by theinvention, as well as antibodies that specifically bind a human c-Mafprotein. The antibodies can be, for example, polyclonal antibodies ormonoclonal antibodies. In one embodiment, the antibodies are coupled toa detectable substance.

Another aspect of the invention pertains to a nonhuman transgenic animalthat contains cells carrying a transgene encoding a human c-Maf protein.

Yet another aspect of the invention pertains to a method for detectingthe presence of human c-Maf in a biological sample. The method involvescontacting the biological sample with an agent capable of detecting anindicator of human c-Maf activity such that the presence of human c-Mafis detected in the biological sample. The invention also provides amethod for modulating human c-Maf activity in a cell comprising,involving contacting the cell with an agent that modulates human c-Mafactivity such that human c-Maf activity in the cell is modulated.

Still another aspect of the invention pertains to methods foridentifying a compound that modulates the activity of a human c-Mafprotein. These methods generally involve:

providing an indicator composition that comprises a human c-Maf protein;

contacting the indicator composition with a test compound; and

determining the effect of the test compound on the activity of the humanc-Maf protein in the indicator composition to thereby identify acompound that modulates the activity of a human c-Maf protein. In apreferred embodiment, the indicator composition comprises a human c-Mafprotein and a DNA molecule to which the human c-Maf protein binds andthe effect of the test compound on the activity of the human c-Mafprotein is determined by evaluating the binding of the human c-Mafprotein to the DNA molecule in the presence and absence of the testcompound. In another preferred embodiment, the indicator composition isa cell comprising a human c-Maf protein and a reporter gene responsiveto the human c-Maf protein and the effect of the test compound on theactivity of the human c-Maf protein is determined by evaluating theexpression of the reporter gene in the presence and absence of the testcompound. In yet another embodiment, the method further involves thestep of determining the effect of the test compound on an immuneresponse to thereby identify a compound that modulates an immuneresponse.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1A-1B is an alignment of the nucleotide sequence of the human c-maf(SEQ ID NO:1) coding region with the mouse c-maf (SEQ ID NO:3) codingregion. Nucleotide differences between the two sequences are boxed.

FIG. 2 is an alignment of the amino acid sequence of the human c-Mafprotein (SEQ ID NO:2) with the mouse c-Maf protein (SEQ ID NO:4). Aminoacid differences between the two sequences are boxed.

DETAILED DESCRIPTION OF THE INVENTION

This invention pertains to human c-Maf compositions, such as isolatednucleic acid molecules encoding human c-Maf and isolated human c-Mafproteins, as well as methods of use therefore. The human compositions ofthe invention have the advantages that they function optimally in humancells (compared with non-human c-Maf compositions) and do not stimulatean immune response in humans.

So that the invention may be more readily understood, certain terms arefirst defined.

As used herein, the term “human c-Maf” is intended to encompass proteinsthat share the distinguishing structural and functional features(described further herein) of the human c-Maf protein encoded by theNh2I/XbaI insert of plasmid pHu-c-Maf, which was deposited under theprovisions of the Budapest Treaty with the American Type Tissue CultureCollection (ATCC), P.O. Box 1549, Manassas, Va. 20108, on Feb. 24, 1998and assigned ATCC Acession No. 98671, and having the amino acid sequenceof SEQ ID NO: 2, including the amino acid residues unique to human c-Maf(as compared to mouse c-Maf), which are boxed in FIG. 2.

As used herein, the term “nucleic acid molecule” is intended to includeDNA molecules (e.g., cDNA or genomic DNA) and RNA molecules (e.g.,mRNA). The nucleic acid molecule may be single-stranded ordouble-stranded, but preferably is double-stranded DNA.

An used herein, an “isolated nucleic acid molecule” refers to a nucleicacid molecule that is free of gene sequences which naturally flank thenucleic acid in the genomic DNA of the organism from which the nucleicacid is derived (i.e., genetic sequences that are located adjacent tothe gene for the isolated nucleic molecule in the genomic DNA of theorganism from which the nucleic acid is derived). For example, invarious embodiments, an isolated human c-Maf nucleic acid moleculetypically contains less than about 10 kb of nucleotide sequences whichnaturally flank the nucleic acid molecule in genomic DNA of the cellfrom which the nucleic acid is derived, and more preferably containsless than about 5, kb, 4 kb, 3 kb, 2 kb, 1 kb, 0.5 kb or 0.1 kb ofnaturally flanking nucleotide sequences. An “isolated” human c-Mafnucleic acid molecule may, however, be linked to other nucleotidesequences that do not normally flank the human c-Maf sequences ingenomic DNA (e.g., the human c-Maf nucleotide sequences may be linked tovector sequences). In certain preferred embodiments, an “isolated”nucleic acid molecule, such as a cDNA molecule, also may be free ofother cellular material. However, it is not necessary for the humanc-Maf nucleic acid molecule to be free of other cellular material to beconsidered “isolated” (e.g., a human c-Maf DNA molecule separated fromother mammalian DNA and inserted into a bacterial cell would still beconsidered to be “isolated”).

As used herein, the term “hybridizes under high stringency conditions”is intended to describe conditions for hybridization and washing underwhich nucleotide sequences having substantial homology (e.g., typicallygreater than 70% homology) to each other remain stably hybridized toeach other. A preferred, non-limiting example of high stringencyconditions are hybridization in a hybridization buffer that contains 6×sodium chloride/sodium citrate (SSC) at a temperature of about 45° C.for several hours to overnight, followed by one or more washes in awashing buffer containing 0.2×SSC, 0.1% SDS at a temperature of about50-65° C.

The term “% identity” as used in the context of nucleotide and aminoacid sequences (e.g., when one amino acid sequence is said to be X %identical to another amino acid sequence) refers to the percentage ofidentical residues shared between the two sequences, when optimallyaligned. To determine the percent identity of two nucleotide or aminoacid sequences, the sequences are aligned for optimal comparisonpurposes (e.g., gaps may be introduced in one sequence for optimalalignment with the other sequence). The residues at correspondingpositions are then compared and when a position in one sequence isoccupied by the same residue as the corresponding position in the othersequence, then the molecules are identical at that position. The percentidentity between two sequences, therefore, is a function of the numberof identical positions shared by two sequences (i.e., % identity=# ofidentical positions/total # of positions×100).

Computer algorithms known in the art can be used to optimally align andcompare two nucleotide or amino acid sequences to define the percentidentity between the two sequences. A preferred, non-limiting example ofa mathematical algorithim utilized for the comparison of two sequencesis the algorithm of Karlin and Altschul (1990) Proc. Natl. Acad. Sci.USA 87:2264-68, modified as in Karlin and Altschul (1993) Proc. Natl.Acad. Sci. USA 90:5873-77. Such an algorithm is incorporated into theNBLAST and XBLAST programs of Altschul, et al. (1990) J. Mol. Biol.215:403-10. To obtain gapped alignments for comparison purposes, GappedBLAST can be utilized as described in Altschul et al., (1997) NucleicAcids Research 25(17):3389-3402. When utilizing BLAST and Gapped BLASTprograms, the default parameters of the respective programs (e.g.,XBLAST and NBLAST) can be used. See http://www.ncbi.nlm.nih.gov. Anotherpreferred, non-limiting example of a mathematical algorithim utilizedfor the comparison of sequences is the algorithm of Myers and Miller,CABIOS (1989). Such an algorithm is incorporated into the ALIGN program(version 2.0) which is part of the GCG sequence alignment softwarepackage. When utilizing the ALIGN program for comparing amino acidsequences, a PAM120 weight residue table, a gap length penalty of 12,and a gap penalty of 4 can be used. If multiple programs are used tocompare sequences, the program that provides optimal alignment (i.e.,the highest percent identity between the two sequences) is used forcomparison purposes.

As used herein, a “naturally-occurring” nucleic acid molecule refers toan RNA or DNA molecule having a nucleotide sequence that occurs innature (e.g., encodes a natural protein).

As used herein, an “antisense” nucleic acid comprises a nucleotidesequence which is complementary to a “sense” nucleic acid encoding aprotein, e.g., complementary to the coding strand of a double-strandedcDNA molecule, complementary to an mRNA sequence or complementary to thecoding strand of a gene. Accordingly, an antisense nucleic acid canhydrogen bond to a sense nucleic acid.

As used herein, the term “coding region” refers to regions of anucleotide sequence comprising codons which are translated into aminoacid residues, whereas the term “noncoding region” refers to regions ofa nucleotide sequence that are not translated into amino acids (e.g., 5′and 3′ untranslated regions).

As used herein, the term “vector” refers to a nucleic acid moleculecapable of transporting another nucleic acid to which it has beenlinked. One type of vector is a “plasmid”, which refers to a circulardouble stranded DNA loop into which additional DNA segments may beligated. Another type of vector is a viral vector, wherein additionalDNA segments may be ligated into the viral genome. Certain vectors arecapable of autonomous replication in a host cell into which they areintroduced (e.g., bacterial vectors having a bacterial origin ofreplication and episomal mammalian vectors). Other vectors (e.g.,non-episomal mammalian vectors) are integrated into the genome of a hostcell upon introduction into the host cell, and thereby are replicatedalong with the host genome. Moreover, certain vectors are capable ofdirecting the expression of genes to which they are operatively linked.Such vectors are referred to herein as “recombinant expression vectors”or simply “expression vectors”. In general, expression vectors ofutility in recombinant DNA techniques are often in the form of plasmids.In the present specification, “plasmid” and “vector” may be usedinterchangeably as the plasmid is the most commonly used form of vector.However, the invention is intended to include such other forms ofexpression vectors, such as viral vectors (e.g., replication defectiveretroviruses, adenoviruses and adeno-associated viruses), which serveequivalent functions.

As used herein, the term “host cell” is intended to refer to a cell intowhich a nucleic acid of the invention, such as a recombinant expressionvector of the invention, has been introduced. The terms “host cell” and“recombinant host cell” are used interchangeably herein. It should beunderstood that such terms refer not only to the particular subject cellbut to the progeny or potential progeny of such a cell. Because certainmodifications may occur in succeeding generations due to either mutationor environmental influences, such progeny may not, in fact, be identicalto the parent cell, but are still included within the scope of the termas used herein.

As used herein, a “transgenic animal” refers to a non-human animal,preferably a mammal, more preferably a mouse, in which one or more ofthe cells of the animal includes a “transgene”. The term “transgene”refers to exogenous DNA which is integrated into the genome of a cellfrom which a transgenic animal develops and which remains in the genomeof the mature animal, for example directing the expression of an encodedgene product in one or more cell types or tissues of the transgenicanimal.

As used herein, a “homologous recombinant animal” refers to a type oftransgenic non-human animal, preferably a mammal, more preferably amouse, in which an endogenous gene has been altered by homologousrecombination between the endogenous gene and an exogenous DNA moleculeintroduced into a cell of the animal, e.g., an embryonic cell of theanimal, prior to development of the animal.

As used herein, an “isolated protein” refers to a protein that issubstantially free of other proteins, cellular material and culturemedium when isolated from cells or produced by recombinant DNAtechniques, or chemical precursors or other chemicals when chemicallysynthesized.

As used herein, the term “antibody” is intended to includeimmunoglobulin molecules and immunologically active portions ofimmunoglobulin molecules, i.e., molecules that contain an antigenbinding site which specifically binds (immunoreacts with) an antigen,such as Fab and F(ab′)₂ fragments. The terms “monoclonal antibodies” and“monoclonal antibody composition”, as used herein, refer to a populationof antibody molecules that contain only one species of an antigenbinding site capable of immunoreacting with a particular epitope of anantigen, whereas the term “polyclonal antibodies” and “polyclonalantibody composition” refer to a population of antibody molecules thatcontain multiple species of antigen binding sites capable of interacingwith a particular antigen. A monoclonal antibody compositions thustypically display a single binding affinity for a particular antigenwith which it immunoreacts.

There is a known and definite correspondence between the amino acidsequence of a particular protein and the nucleotide sequences that cancode for the protein, as defined by the genetic code (shown below).Likewise, there is a known and definite correspondence between thenucleotide sequence of a particular nucleic acid molecule and the aminoacid sequence encoded by that nucleic acid molecule, as defined by thegenetic code.

GENETIC CODE Alanine (Ala, A) GCA, GCC, GCG, GCT Arginine (Arg, R) AGA,ACG, CGA, CGC, CGG, CGT Asparagine (Asn, N) AAC, AAT Aspartic acid(Asp,D) GAC, GAT Cysteine (Cys, C) TGC, TGT Glutamic acid (Glu,E) GAA,GAG Glutamine (Gln, Q) CAA, CAG Glycine (Gly, G) GGA, GGC, GGG, GGTHistidine (His, H) CAC, CAT Isoleucine (Ile, I) ATA, ATC, ATT Leucine(Leu, L) CTA, CTC, CTG, CTT, TTA, TTG Lysine (Lys, K) AAA, AAGMethionine (Met, M) ATG Phenylalanine (Phe,F) TTC, TTT Proline (Pro, P)CCA, CCC, CCG, CCT Serine (Ser, S) AGC, AGT, TCA, TCC, TCG, TCTThreonine (Thr, T) ACA, ACC, ACG, ACT Tryptophan (Trp, W) TGG Tyrosine(Tyr, Y) TAC, TAT Valine (Val, V) GTA, GTC, GTG, GTT Termination signal(end) TAA, TAG, TGAAn important and well known feature of the genetic code is itsredundancy, whereby, for most of the amino acids used to make proteins,more than one coding nucleotide triplet may be employed (illustratedabove). Therefore, a number of different nucleotide sequences may codefor a given amino acid sequence. Such nucleotide sequences areconsidered functionally equivalent since they result in the productionof the same amino acid sequence in all organisms (although certainorganisms may translate some sequences more efficiently than they doothers). Moreover, occasionally, a methylated variant of a purine orpyrimidine may be found in a given nucleotide sequence. Suchmethylations do not affect the coding relationship between thetrinucleotide codon and the corresponding amino acid.

In view of the foregoing, the nucleotide sequence of a DNA or RNAmolecule coding for a human c-Maf protein of the invention (or anyportion thereof) can be use to derive the human c-Maf amino acidsequence, using the genetic code to translate the DNA or RNA moleculeinto an amino acid sequence. Likewise, for any human c-Maf-amino acidsequence, corresponding nucleotide sequences that can encode the humanc-Maf protein can be deduced from the genetic code (which, because ofits redundancy, will produce multiple nucleic acid sequences for anygiven amino acid sequence). Thus, description and/or disclosure hereinof a human c-Maf nucleotide sequence should be considered to alsoinclude description and/or disclosure of the amino acid sequence encodedby the nucleotide sequence. Similarly, description and/or disclosure ofa human c-Maf amino acid sequence herein should be considered to alsoinclude description and/or disclosure of all possible nucleotidesequences that can encode the amino acid sequence.

Various aspects of the invention are described in further detail in thefollowing subsections:

I. Isolated Nucleic Acid Molecules

One aspect of the invention pertains to isolated nucleic acid moleculesthat encode human c-Maf. An approximately 4.2 kilobase fragment of DNAencoding human c-Maf has been isolated from a genomic DNA library andsubcloned into the plasmid pBluescriptKS/II. E. coli bacteria carryingthis plasmid, referred to as pHu-c-Maf, have been deposited under theprovisions of the Budapest Treaty with the American Type CultureCollection (ATCC), P.O. Box 1549, Manassas, Va. 20108, on Feb. 24, 1998and assigned ATCC Accession No. 98671. This plasmid was constructed byinsertion of a ˜4.2 kb NheI fragment encompassing the human c-Maf codingregion into the compatible XbaI site of the plasmid vector, to therebycreate a ˜4.2 kb NheI/XbaI insert that encodes human c-Maf. It should benoted that upon ligation of the NheI fragment into the XbaI site, theserestriction sites are not regenerated and, thus, to excise the fragmentfrom the plasmid, it is necessary to use adjacent restriction siteswithin the pBluescript polylinker. The nucleotide sequence of the humanc-Maf coding region, and corresponding predicted amino acid sequence,are shown in SEQ ID NOs: 1 and 2, respectively. This nucleotidesequence, and predicted amino acid sequence, of human c-Maf wereobtained by sequencing of the NheI/XbaI insert of the pHu-c-Maf plasmidusing standard sequencing methods. Primers for sequencing are designedbased on the nucleotide sequence shown in SEQ ID NO: 1. Isolation andcharacterization of the human c-Maf-encoding DNA is described further inthe Example.

In a preferred embodiment, the nucleic acid molecule of the inventioncomprises the nucleotide sequence of the coding region of the NheI/XbaIinsert of plasmid pHu-c-Maf (ATCC Accession No. 98671). In anotherpreferred embodiment, the nucleic acid moleculecomprises the nucleotidesequence of SEQ ID NO: 1. In other embodiments, the nucleic acidmolecule has at least 98% nucleotide identity, more preferably 99%nucleotide identity, and even more preferably 99.5% nucleotide identitywith the nucleotide sequence of SEQ ID NO: 1 or the nucleotide sequenceof the NheI/XbaI insert of plasmid pHu-c-Maf (ATCC Accession No. 98671).In yet another embodiment, the nucleic acid molecule comprising thenucleotide sequence of the NheI/XbaI insert of plasmid pHu-c-Maf (ATCCAccession No. 98671).

Nucleic acid molecules that differ from SEQ ID NO: 1 (and nucleotidesequence of the NheI/XbaI insert of p-Hu-c-Maf) due to degeneracy of thegenetic code, and thus encode the same human c-Maf protein as thatencoded by SEQ ID NO: 1 and pHu-c-Maf, are encompassed by the invention.Accordingly, in another embodiment, an isolated nucleic acid molecule ofthe invention has a nucleotide sequence encoding a protein having anamino acid sequence shown in SEQ ID NO: 2 or having the amino acidsequence encoded by the coding region of the NheI/XbaI insert ofp-Hu-c-Maf.

A nucleic acid molecule having the nucleotide sequence of human c-Mafcan be obtained from plasmid pHu-c-Maf or can be isolated using standardmolecular biology techniques and the sequence information providedherein. For example, a human c-Maf DNA can be isolated from a humangenomic DNA library using all or portion of SEQ ID NO: 1 as ahybridization probe and standard hybridization techniques (e.g., asdescribed in Sambrook, J., et al. Molecular Cloning: A LaboratoryManual. 2nd, ed., Cold Spring Harbor Laboratory, Cold Spring Harbor,N.Y., 1989). Moreover, a nucleic acid molecule encompassing all or aportion of SEQ ID NO: 1 can be isolated by the polymerase chain reactionusing oligonucleotide primers designed based upon the sequence of SEQ IDNO: 1. For example, mRNA can be isolated from cells (e.g., by theguanidinium-thiocyanate extraction procedure of Chirgwin et al. (1979)Biochemistry 18: 5294-5299) and cDNA can be prepared using reversetranscriptase (e.g., Moloney MLV reverse transcriptase, available fromGibco/BRL, Bethesda, Md.; or AMV reverse transcriptase, available fromSeikagaku America, Inc., St. Petersburg, Fla.). Syntheticoligonucleotide primers for PCR amplification can be designed based uponthe nucleotide sequence shown in SEQ ID NO: 1. A nucleic acid of theinvention can be amplified using cDNA or, alternatively, genomic DNA, asa template and appropriate oligonucleotide primers according to standardPCR amplification techniques. The nucleic acid so amplified can becloned into an appropriate vector and characterized by DNA sequenceanalysis. Furthermore, oligonucleotides corresponding to a human c-Mafnucleotide sequence can be prepared by standard synthetic techniques,e.g., using an automated DNA synthesizer.

In addition to the human c-Maf nucleotide sequence shown in SEQ ID NO: 1and carried by plasmid pHu-c-Maf, it will be appreciated by thoseskilled in the art that DNA sequence polymorphisms that lead to minorchanges in the nucleotide or amino acid sequences of human c-Maf mayexist within a population. Such genetic polymorphism in the human c-Mafgene may exist among individuals within a population due to naturalallelic variation. Such natural allelic variations can typically resultin 1-2% variance in the nucleotide sequence of the a gene. Any and allsuch nucleotide variations and resulting amino acid polymorphisms inhuman c-Maf that are the result of natural allelic variation and that donot alter the functional activity of human c-Maf are intended to bewithin the scope of the invention.

Nucleic acid molecules corresponding to natural allelic variants of thehuman c-Maf DNAs of the invention can be isolated based on theirhomology to the human c-Maf nucleic acid molecules disclosed hereinusing the human DNA, or a portion thereof, as a hybridization probeaccording to standard hybridization techniques under high stringencyhybridization conditions. Accordingly, in another embodiment, anisolated nucleic acid molecule of the invention hybridizes under highstringency conditions to a second nucleic acid molecule comprising thenucleotide sequence of SEQ ID NO: 1. In certain embodiment, the isolatednucleic acid molecule comprises at least 30, 50, 100, 200, 300, 400,500, 600, 700, 800, 900, 1000, 2000 or 3000 contiguous nucleotides ofSEQ ID NO: 1. Preferably, an isolated nucleic acid molecule of theinvention that hybridizes under high stringency conditions to thesequence of SEQ ID NO: 1 corresponds to a naturally-occurring allelicvariant of a human c-Maf nucleic acid molecule.

In addition to naturally-occurring allelic variants of the human c-Mafsequence that may exist in the population, the skilled artisan willfurther appreciate that minor changes may be introduced by mutation intothe nucleotide sequence of SEQ ID NO: 1, thereby leading to changes inthe amino acid sequence of the encoded protein, without altering thefunctional activity of the human c-Maf protein. For example, nucleotidesubstitutions leading to amino acid substitutions at “non-essential”amino acid residues may be made in the sequence of SEQ ID NO: 1. A“non-essential” amino acid residue is a residue that can be altered fromthe wild-type sequence of human c-Maf (e.g., the sequence of SEQ ID NO:2) without altering the functional activity of c-Maf, such as itsability to interact with DNA or its ability to enhance transcriptionfrom an IL-4 promoters whereas an “essential” amino acid residue isrequired for functional activity.

Accordingly, another aspect of the invention pertains to nucleic acidmolecules encoding human c-Maf proteins that contain changes in aminoacid residues that are not essential for human c-Maf activity. Suchhuman c-Maf proteins differ in amino acid sequence from SEQ ID NO: 2 (orthe amino acid sequence encoded by pHu-c-Maf) yet retain human c-Mafactivity. These non-natural variants of human c-Maf also differ fromnon-human c-Maf proteins (e.g., chicken or mouse c-Maf) in that theyencode at least one amino acid residue that is unique to human c-Maf(i.e., at least one residue that is not present in chicken or mousec-Maf). Preferably, these non-natural variants of human c-Maf encode atleast 2, 3, 4, 5, 6, 7, 8, 9 or 10 amino acid residues that are uniqueto human c-Maf (i.e., that are not present in chicken or mouse c-Maf).

An isolated nucleic acid molecule encoding a non-natural variant of ahuman c-Maf protein can be created by introducing one or more nucleotidesubstitutions, additions or deletions into the nucleotide sequence ofSEQ ID NO: 1 (or plasmid pHu-c-Maf) such that one or more amino acidsubstitutions, additions or deletions are introduced into the encodedprotein. Mutations can be introduced into SEQ ID NO: 1 by standardtechniques, such as site-directed mutagenesis and PCR-mediatedmutagenesis. Preferably, conservative amino acid substitutions are madeat one or more non-essential amino acid residues. A “conservative aminoacid substitution” is one in which the amino acid residue is replacedwith an amino acid residue having a similar side chain. Families ofamino acid residues having similar side chains have been defined in theart, including basic side chains (e.g., lysine, arginine, histidine),acidic side chains (e.g., aspartic acid, glutamic acid), uncharged polarside chains (e.g., glycine, asparagine, glutamine, serine, threonine,tyrosine, cysteine), nonpolar side chains (e.g., alanine, valine,leucine, isoleucine, proline, phenylalanine, methionine, tryptophan),beta-branched side chains (e.g., threonine, valine, isoleucine) andaromatic side chains (e.g., tyrosine, phenylalanine, tryptophan,histidine). Thus, a nonessential amino acid residue in human c-Maf ispreferably replaced with another amino acid residue from the same sidechain family. Alternatively, in another embodiment, mutations can beintroduced randomly along all or part of the human c-Maf codingsequence, such as by saturation mutagenesis, and the resultant mutantscan be screened for their ability to bind to DNA and/or activatetranscription, to identify mutants that retain functional activity.Following mutagenesis, the encoded human c-Maf mutant protein can beexpressed recombinantly in a host cell and the functional activity ofthe mutant protein can be determined using assays available in the artfor assessing c-Maf activity (e.g., assays such as those described indetail in PCT Publication WO 97/39721.

Another aspect of the invention pertains to isolated nucleic acidmolecules that are antisense to the coding strand of a human c-Maf mRNAor gene. An antisense nucleic acid of the invention can be complementaryto an entire human c-Maf coding strand, or to only a portion thereof. Inone embodiment, an antisense nucleic acid molecule is antisense to acoding region of the coding strand of a nucleotide sequence encodinghuman c-Maf that is unique to human c-Maf (as compared to non-humanc-Mafs, such as chicken or mouse c-Maf). In another embodiment, theantisense nucleic acid molecule is antisense to a noncoding region ofthe coding strand of a nucleotide sequence encoding human c-Maf that isunique to human c-Maf (as compared to non-human c-Mafs, such as chickenor mouse c-Maf). In preferred embodiments, an antisense of the inventioncomprises at least 30 contiguous nucleotides of the noncoding strand ofSEQ ID NO: 1, more preferably at least 50, 100, 200, 300, 400, 500, 600,700, 800, 900 or 1000 contiguous nucleotides of the noncoding strand ofSEQ ID NO: 1.

Given the coding strand sequences encoding human c-Maf disclosed herein(e.g., SEQ ID NO: 1 and plasmid pHu-c-Maf), antisense nucleic acids ofthe invention can be designed according to the rules of Watson and Crickbase pairing. The antisense nucleic acid molecule may be complementaryto the entire coding region of human c-Maf mRNA, or alternatively can bean oligonucleotide which is antisense to only a portion of the coding ornoncoding region of human c-Maf mRNA. For example, the antisenseoligonucleotide may be complementary to the region surrounding thetranslation start site of human c-Maf mRNA. An antisense oligonucleotidecan be, for example, about 15, 20, 25, 30, 35, 40, 45 or 50 nucleotidesin length. An antisense nucleic acid of the invention can be constructedusing chemical synthesis and enzymatic ligation reactions usingprocedures known in the art. For example, an antisense nucleic acid(e.g., an antisense oligonucleotide) can be chemically synthesized usingnaturally occurring nucleotides or variously modified nucleotidesdesigned to increase the biological stability of the molecules or toincrease the physical stability of the duplex formed between theantisense and sense nucleic acids, e.g., phosphorothioate derivativesand acridine substituted nucleotides can be used. Alternatively, theantisense nucleic acid can be produced biologically using an expressionvector into which a nucleic acid has been subcloned in an antisenseorientation (i.e., RNA transcribed from the inserted nucleic acid willbe of an antisense orientation to a target nucleic acid of interest,described further in the following subsection).

In another embodiment, an antisense nucleic acid of the invention is aribozyme. Ribozymes are catalytic RNA molecules with ribonucleaseactivity which are capable of cleaving a single-stranded nucleic acid,such as an mRNA, to which they have a complementary region. A ribozymehaving specificity for a human c-Maf-encoding nucleic acid can bedesigned based upon the nucleotide sequence of a human c-Maf genedisclosed herein. For example, a derivative of a Tetrahymena L-19 IVSRNA can be constructed in which the base sequence of the active site iscomplementary to the base sequence to be cleaved in a humanc-Maf-encoding mRNA. See for example Cech et al. U.S. Pat. No.4,987,071; and Cech et al. U.S. Pat. No. 5,116,742. Alternatively, humanc-Maf mRNA can be used to select a catalytic RNA having a specificribonuclease activity from a pool of RNA molecules. See for exampleBartel, D. and Szostak, J. W. (1993) Science 261: 1411-1418.

Yet another aspect of the invention pertains to isolated nucleic acidmolecules encoding human c-Maf fusion proteins. Such nucleic acidmolecules, comprising at least a first nucleotide sequence encoding ahuman c-Maf protein, polypeptide or peptide operatively linked to asecond nucleotide sequence encoding a non-human c-Maf protein,polypeptide or peptide, can be prepared by standard recombinant DNAtechniques. Human c-Maf fusion proteins are described in further detailbelow in subsection III.

II. Recombinant Expression Vectors and Host Cells

Another aspect of the invention pertains to vectors, preferablyrecombinant expression vectors, containing a nucleic acid encoding humanc-Maf (or a portion thereof). The expression vectors of the inventioncomprise a nucleic acid of the invention in a form suitable forexpression of the nucleic acid in a host cell, which means that therecombinant expression vectors include one or more regulatory sequences,selected on the basis of the host cells to be used for expression, whichis operatively linked to the nucleic acid sequence to be expressed.Within a recombinant expression vector, “operably linked” is intended tomean that the nucleotide sequence of interest is linked to theregulatory sequence(s) in a manner which allows for expression of thenucleotide sequence (e.g., in an in vitro transcription/translationsystem or in a host cell when the vector is introduced into the hostcell). The term “regulatory sequence” is intended to includes promoters,enhancers and other expression control elements (e.g., polyadenylationsignals). Such regulatory sequences are described, for example, inGoeddel; Gene Expression Technology: Methods in Enzymology 185, AcademicPress, San Diego, Calif. (1990). Regulatory sequences include thosewhich direct constitutive expression of a nucleotide sequence in manytypes of host cell and those which direct expression of the nucleotidesequence only in certain host cells (e.g., tissue-specific regulatorysequences). It will be appreciated by those skilled in the art that thedesign of the expression vector may depend on such factors as the choiceof the host cell to be transformed, the level of expression of proteindesired, etc. The expression vectors of the invention can be introducedinto host cells to thereby produce proteins or peptides, includingfusion proteins or peptides, encoded by nucleic acids as describedherein (e.g., human c-Maf proteins, mutant forms of human c-Mafproteins, human c-Maf fusion proteins and the like).

The recombinant expression vectors of the invention can be designed forexpression of human c-Maf protein in prokaryotic or eukaryotic cells.For example, human c-Maf can be expressed in bacterial cells such as E.coli, insect cells (using baculovirus expression vectors) yeast cells ormammalian cells. Suitable host cells are discussed further in Goeddel,Gene Expression Technology: Methods in Enzymology 185, Academic Press,San Diego, Calif. (1990). Alternatively, the recombinant expressionvector may be transcribed and translated in vitro, for example using T7promoter regulatory sequences and T7 polymerase.

Expression of proteins in prokaryotes is most often carried out in E.coli with vectors containing constitutive or inducible promotorsdirecting the expression of either fusion or non-fusion proteins. Fusionvectors add a number of amino acids to a protein encoded therein,usually to the amino terminus of the recombinant protein. Such fusionvectors can serve one or more purposes: 1) to increase expression ofrecombinant protein; 2) to increase the solubility of the recombinantprotein; 3) to aid in the purification of the recombinant protein byacting as a ligand in affinity purification; 4) to provide an epitopetag to aid in detection and/or purification of the protein; and/or 5) toprovide a marker to aid in detection of the protein (e.g., a colormarker using β-galactosidase fusions). Often, in fusion expressionvectors, a proteolytic cleavage site is introduced at the junction ofthe fusion moiety and the recombinant protein to enable separation ofthe recombinant protein from the fusion moiety subsequent topurification of the fusion protein. Such enzymes, and their cognaterecognition sequences, include Factor Xa, thrombin and enterokinase.Typical fusion expression vectors include pGEX (Pharmacia Biotech Inc.;Smith, D. B. and Johnson, K. S. (1988) Gene 67:31-40), pMAL (New EnglandBiolabs, Beverly, Mass.) and pRIT5 (Pharmacia, Piscataway, N.J.) whichfuse glutathione S-transferase (GST), maltose E binding protein, orprotein A, respectively, to the target recombinant protein. Recombinantproteins also can be expressed in eukaryotic cells as fusion proteinsfor the same purposes discussed above.

Examples of suitable inducible non-fusion E. coli expression vectorsinclude pTrc (Amann et al., (1988) Gene 69:301-315) and pET 11d (Studieret al., Gene Expression Technology: Methods in Enzymology 185, AcademicPress, San Diego, Calif. (1990) 60-89). Target gene expression from thepTrc vector relies on host RNA polymerase transcription from a hybridtrp-lac fusion promoter. Target gene expression from the pET 11d vectorrelies on transcription from a T7 gn10-lac fusion promoter mediated by acoexpressed viral RNA polymerase (T7 gn1). This viral polymerase issupplied by host strains BL21(DE3) or HMS174(DE3) from a resident λprophage harboring a T7 gn1 gene under the transcriptional control ofthe lacUV 5 promoter.

One strategy to maximize recombinant protein expression in E. coli is toexpress the protein in a host bacteria with an impaired capacity toproteolytically cleave the recombinant protein (Gottesman, S., GeneExpression Technology: Methods in Enzymology 185, Academic Press, SanDiego, Calif. (1990) 119-128). Another strategy is to alter the nucleicacid sequence of the nucleic acid to be inserted into an expressionvector so that the individual codons for each amino acid are thosepreferentially utilized in E. coli (Wada et al., (1992) Nuc. Acids Res.20:2111-2118). Such alteration of nucleic acid sequences of theinvention can be carried out by standard DNA synthesis techniques.

In another embodiment, the human c-Maf expression vector is a yeastexpression vector. Examples of vectors for expression in yeast S.cerivisae include pYepSec1 (Baldari. et al., (1987) EMBO J. 6:229-234),pMFa (Kurjan and Herskowitz, (1982) Cell 30:933-943), pJRY88 (Schultz etal, (1987) Gene 54:113-123), and pYES2 (Invitrogen Corporation, SanDiego, Calif.).

Alternatively, human c-Maf can be expressed in insect cells usingbaculovirus expression vectors. Baculovirus vectors available forexpression of proteins in cultured insect cells (e.g., Sf9 cells)include the pAc series (Smith et al., (1983) Mol. Cell Biol.3:2156-2165) and the pVL series (Lucklow, V. A., and Summers, M. D.,(1989) Virology 170:31-39).

In yet another embodiment, a nucleic acid of the invention is expressedin mammalian cells using a mammalian expression vector. Examples ofmammalian expression vectors include pMex-NeoI, pCDM8 (Seed, B., (1987)Nature 329:840) and pMT2PC (Kaufman et al. (1987), EMBO J. 6:187-195).When used in mammalian cells, the expression vector's control functionsare often provided by viral regulatory elements. For example, commonlyused promoters are derived from polyoma, Adenovirus 2, cytomegalovirusand Simian Virus 40.

In another embodiment, the recombinant mammalian expression vector iscapable of directing expression of the nucleic acid preferentially in aparticular cell type (e.g., tissue-specific regulatory elements are usedto express the nucleic acid). Tissue-specific regulatory elements areknown in the art. Non-limiting examples of suitable tissue-specificpromoters include lymphoid-specific promoters (Calame and Eaton (1988)Adv. Immunol. 43:235-275), in particular promoters of T cell receptors(Winoto and Baltimore (1989) EMBO J. 8:729-733) and immunoglobulins(Banerji et al. (1983) Cell 33:729-740; Queen and Baltimore (1983) Cell33:741-748), the albumin promoter (liver-specific; Pinkert et al. (1987)Genes Dev. 1:268-277), neuron-specific promoters (e.g., theneurofilament promoter; Byrne and Ruddle (1989) Proc. Natl. Acad. Sci.USA 86:5473-5477), pancreas-specific promoters (Edlund et al. (1985)Science 230:912-916), and mammary gland-specific promoters (e.g., milkwhey promoter; U.S. Pat. No. 4,873,316 and European ApplicationPublication No. 264,166). Developmentally-regulated promoters are alsoencompassed, for example the murine hox promoters (Kessel and Gruss(1990) Science 249:374-379) and the α-fetoprotein promoter (Campes andTilghman (1989) Genes Dev. 3:537-546).

Moreover, inducible regulatory systems for use in mammalian cells areknown in the art, for example systems in which gene expression isregulated by heavy metal ions (see e.g., Mayo et al. (1982) Cell29:99-108; Brinster et al. (1982) Nature 296:39-42; Searle et al. (1985)Mol. Cell. Biol. 5:1480-1489), heat shock (see e.g., Nouer et al. (1991)in Heat Shock Response, e.d. Nouer, L., CRC, Boca Raton, Fla.,pp167-220), hormones (see e.g., Lee et al. (1981) Nature 294:228-232;Hynes et al. (1981) Proc. Natl. Acad. Sci. USA 78:2038-2042; Klock etal. (1987) Nature 329:734-736; Israel & Kaufman (1989) Nucl. Acids Res.17:2589-2604; and PCT Publication No. WO 93/23431), FK506-relatedmolecules (see e.g., PCT Publication No. WO 94/18317) or tetracyclines(Gossen, M. and Bujard, H. (1992) Proc. Natl. Acad. Sci. USA89:5547-5551; Gossen, M. et al. (1995) Science 268:1766-1769; PCTPublication No. WO 94/29442; and PCT Publication No. WO 96/01313).Accordingly, in another embodiment, the invention provides a recombinantexpression vector in which human c-Maf DNA is operatively linked to aninducible eukaryotic promoter, thereby allowing for inducible expressionof human c-Maf protein in eukaryotic cells.

The invention further provides a recombinant expression vectorcomprising a DNA molecule of the invention cloned into the expressionvector in an antisense orientation. That is, the DNA molecule isoperatively linked to a regulatory sequence in a manner which allows forexpression (by transcription of the DNA molecule) of an RNA moleculewhich is antisense to human c-Maf mRNA. Regulatory sequences operativelylinked to a nucleic acid cloned in the antisense orientation can bechosen which direct the continuous expression of the antisense RNAmolecule in a variety of cell types, for instance viral promoters and/orenhancers, or regulatory sequences can be chosen which directconstitutive, tissue specific or cell type specific expression ofantisense RNA. The antisense expression vector can be in the form of arecombinant plasmid, phagemid or attenuated virus in which antisensenucleic acids are produced under the control of a high efficiencyregulatory region, the activity of which can be determined by the celltype into which the vector is introduced. For a discussion of theregulation of gene expression using antisense genes see Weintraub, H. etal., Antisense RNA as a molecular tool for genetic analysis,Reviews—Trends in Genetics, Vol. 1(1) 1986.

Another aspect of the invention pertains to recombinant host cells intowhich a vector, preferably a recombinant expression vector, of theinvention has been introduced. A host cell may be any prokaryotic oreukaryotic cell. For example, human c-Maf protein may be expressed inbacterial cells such as E. coli, insect cells, yeast or mammalian cells(such as Chinese hamster ovary cells (CHO) or COS cells). Other suitablehost cells are known to those skilled in the art. Vector DNA can beintroduced into prokaryotic or eukaryotic cells via conventionaltransformation or transfection techniques. As used herein, the terms“transformation” and “transfection” are intended to refer to a varietyof art-recognized techniques for introducing foreign nucleic acid (e.g.,DNA) into a host cell, including calcium phosphate or calcium chlorideco-precipitation, DEAE-dextran-mediated transfection, lipofection, orelectroporation. Suitable methods for transforming or transfecting hostcells can be found in Sambrook et al. (Molecular Cloning: A LaboratoryManual, 2nd Edition, Cold Spring Harbor Laboratory press (1989)), andother laboratory manuals.

For stable transfection of mammalian cells, it is known that, dependingupon the expression vector and transfection technique used, only a smallfraction of cells may integrate the foreign DNA into their genome. Inorder to identify and select these integrants, a gene that encodes aselectable marker (e.g., resistance to antibiotics) is generallyintroduced into the host cells along with the gene of interest.Preferred selectable markers include those which confer resistance todrugs, such as G418, hygromycin and methotrexate. Nucleic acid encodinga selectable marker may be introduced into a host cell on the samevector as that encoding human c-Maf or may be introduced on a separatevector. Cells stably transfected with the introduced nucleic acid can beidentified by drug selection (e.g., cells that have incorporated theselectable marker gene will survive, while the other cells die).

A host cell of the invention, such as a prokaryotic or eukaryotic hostcell in culture, can be used to produce (i.e., express) human c-Mafprotein. Accordingly, the invention further provides methods forproducing human c-Maf protein using the host cells of the invention. Inone embodiment, the method comprises culturing the host cell ofinvention (into which a recombinant expression vector encoding humanc-Maf has been introduced) in a suitable medium until human c-Maf isproduced. In another embodiment, the method further comprises isolatinghuman c-Maf from the medium or the host cell. In its native form thehuman c-Maf protein is an intracellular protein and, accordingly,recombinant human c-Maf protein can be expressed intracellularly in arecombinant host cell and then isolated from the host cell, e.g., bylysing the host cell and recovering the recombinant human c-Maf proteinfrom the lysate. Alternatively, recombinant human c-Maf protein can beprepared as a extracellular protein by operatively linking aheterologous signal sequence to the amino-terminus of the protein suchthat the protein is secreted from the host cells. In this case,recombinant human c-Maf protein can be recovered from the culture mediumin which the cells are cultured.

Certain host cells of the invention can also be used to produce nonhumantransgenic animals. For example, in one embodiment, a host cell of theinvention is a fertilized oocyte or an embryonic stem cell into whichhuman c-Maf-coding sequences have been introduced. Such host cells canthen be used to create non-human transgenic animals in which exogenoushuman c-Maf sequences have been introduced into their genome orhomologous recombinant animals in which endogenous c-Maf sequences havebeen altered. Such animals are useful for studying the function and/oractivity of human c-Maf and for identifying and/or evaluating modulatorsof human c-Maf activity. Accordingly, another aspect of the inventionpertains to nonhuman transgenic animals which contain cells carrying atransgene encoding a human c-Maf protein or a portion of a human c-Mafprotein. In a subembodiment, of the transgenic animals of the invention,the transgene alters an endogenous gene encoding an endogenous c-Mafprotein (e.g., homologous recombinant animals in which the endogenousc-Maf gene has been functionally disrupted or “knocked out”, or thenucleotide sequence of the endogenous c-Maf gene has been mutated or thetranscriptional regulatory region of the endogenous c-Maf gene has beenaltered).

A transgenic animal of the invention can be created by introducing humanc-Maf-encoding nucleic acid into the male pronuclei of a fertilizedoocyte, e.g., by microinjection, and allowing the oocyte to develop in apseudopregnant female foster animal. The human c-Maf nucleotide sequenceof SEQ ID NO: 1 (and plasmid pHu-c-Maf) can be introduced as a transgeneinto the genome of a non-human animal. Intronic sequences andpolyadenylation signals can also be included in the transgene toincrease the efficiency of expression of the transgene. Atissue-specific regulatory sequence(s) can be operably linked to thehuman c-Maf transgene to direct expression of human c-Maf protein toparticular cells. Methods for generating transgenic animals via embryomanipulation and microinjection, particularly animals such as mice, havebecome conventional in the art and are described, for example, in U.S.Pat. Nos. 4,736,866 and 4,870,009, both by Leder et al., U.S. Pat. No.4,873,191 by Wagner et al. and in Hogan, B., Manipulating the MouseEmbryo, (Cold Spring Harbor Laboratory Press, Cold Spring Harbor, N.Y.,1986). Similar methods are used for production of other transgenicanimals. A transgenic founder animal can be identified based upon thepresence of the human c-Maf transgene in its genome and/or expression ofhuman c-Maf mRNA in tissues or cells of the animals. A transgenicfounder animal can then be used to breed additional animals carrying thetransgene. Moreover, transgenic animals carrying a transgene encodinghuman c-Maf can further be bred to other transgenic animals carryingother transgenes.

To create a homologous recombinant animal, a vector is prepared whichcontains at least a portion of a human c-Maf gene into which a deletion,addition or substitution has been introduced to thereby alter, e.g.,functionally disrupt, the endogenous c-Maf gene. In one embodiment, ahomologous recombination vector is designed such that, upon homologousrecombination, the endogenous c-Maf gene is functionally disrupted(i.e., no longer encodes a functional protein; also referred to as a“knock out” vector). Alternatively, the vector can be designed suchthat, upon homologous recombination, the endogenous c-Maf gene replacedby the human c-Maf gene. In the homologous recombination vector, thealtered portion of the c-Maf gene is flanked at its 5′ and 3′ ends byadditional nucleic acid of the c-Maf gene to allow for homologousrecombination to occur between the exogenous human c-Maf gene carried bythe vector and an endogenous c-Maf gene in an embryonic stem cell. Theadditional flanking c-Maf nucleic acid is of sufficient length forsuccessful homologous recombination with the endogenous gene. Typically,several kilobases of flanking DNA (both at the 5′ and 3′ ends) areincluded in the vector (see e.g., Thomas, K. R. and Capecchi, M. R.(1987) Cell 51:503 for a description of homologous recombinationvectors). The vector is introduced into an embryonic stem cell line(e.g., by electroporation) and cells in which the introduced human c-Mafgene has homologously recombined with the endogenous c-Maf gene areselected (see e.g., Li, E. et al. (1992) Cell 69:915). The selectedcells are then injected into a blastocyst of an animal (e.g., a mouse)to form aggregation chimeras (see e.g., Bradley, A. in Teratocarcinomasand Embryonic Stem Cells: A Practical Approach, E. J. Robertson, ed.(IRL, Oxford, 1987) pp. 113-152). A chimeric embryo can then beimplanted into a suitable pseudopregnant female foster animal and theembryo brought to term. Progeny harboring the homologously recombinedDNA in their germ cells can be used to breed animals in which all cellsof the animal contain the homologously recombined DNA by germlinetransmission of the transgene. Methods for constructing homologousrecombination vectors and homologous recombinant animals are describedfurther in Bradley, A. (1991) Current Opinion in Biotechnology 2:823-829and in PCT International Publication Nos.: WO 90/11354 by Le Mouellec etal.; WO 91/01140 by Smithies et al.; WO 92/0968 by Zijlstra et al.; andWO 93/04169 by Berns et al.

In addition to the foregoing, the skilled artisan will appreciate thatother approaches known in the art for homologous recombination can beapplied to the instant invention. Enzyme-assisted site-specificintegration systems are known in the art and can be applied to integratea DNA molecule at a predetermined location in a second target DNAmolecule. Examples of such enzyme-assisted integration systems includethe Cre recombinase-lox target system (e.g., as described in Baubonis,W. and Sauer, B. (1993) Nucl. Acids Res. 21:2025-2029; and Fukushige, S.and Sauer, B. (1992) Proc. Natl. Acad. Sci. USA 89:7905-7909) and theFLP recombinase-FRT target system (e.g., as described in Dang, D. T. andPerrimon, N. (1992) Dev. Genet. 13:367-375; and Fiering, S. et al.(1993) Proc. Natl. Acad. Sci. USA 90:8469-8473). Tetracycline-regulatedinducible homologous recombination systems, such as described in PCTPublication No. WO 94/29442 and PCT Publication No. WO 96/01313, alsocan be used.

III. Isolated Human c-Maf Proteins and Anti-Human c-Maf Antibodies

Another aspect of the invention pertains to isolated human c-Mafproteins. Preferably, the human c-Maf protein comprises the amino acidsequence encoded by the coding region of the NheI/XbaI insert of plasmidpHu-c-Maf (ATCC Accession No. 98671). In another preferred embodiment,the protein comprises the amino acid sequence of SEQ ID NO: 2. In otherembodiments, the protein has at least 98% amino acid identity, morepreferably 99% amino identity, and even more preferably 99.5% amino acididentity with SEQ ID NO: 2 or the protein encoded by the coding regionof the NheI/XbaI insert of plasmid pHu-c-Maf (ATCC Accession No. 98671).

In other embodiments, the invention provides isolated portions of thehuman c-Maf protein. For example, the invention further encompasses anamino-terminal portion of human c-Maf that includes a transcriptionalactivation domain. In various embodiments, this amino terminal portionencompasses at least amino acids 1-122, at least amino acids 1-187, orat least amino acids 1-257. Another isolated portion of human c-Mafprovided by the invention is a portion encompassing a carboxy-terminalleucine zipper domain. This portion encompasses at least amino acids313-348.

Human c-Maf proteins of the invention are preferably produced byrecombinant DNA techniques. For example, a nucleic acid moleculeencoding the protein is cloned into an expression vector (as describedabove), the expression vector is introduced into a host cell (asdescribed above) and the human c-Maf protein is expressed in the hostcell. The human c-Maf protein can then be isolated from the cells by anappropriate purification scheme using standard protein purificationtechniques. Alternative to recombinant expression, a human c-Mafpolypeptide can be synthesized chemically using standard peptidesynthesis techniques. Moreover, native human c-Maf protein can beisolated from cells (e.g., from T cells), for example byimmunoprecipitation using an anti-human c-Maf antibody.

The invention also provides human c-Maf fusion proteins. As used herein,a human c-Maf “fusion protein” comprises a human c-Maf polypeptideoperatively linked to a polypeptide other than human c-Maf. A “humanc-Maf polypeptide” refers to a polypeptide having an amino acid sequencecorresponding to human c-Maf protein, or a peptide fragment thereofwhich is unique to human c-Maf protein (as compared to non-human c-Mafproteins, such as mouse or chicken c-Maf”, whereas a “polypeptide otherthan human c-Maf” refers to a polypeptide having an amino acid sequencecorresponding to another protein. Within the fusion protein, the term“operatively linked” is intended to indicate that the human c-Mafpolypeptide and the other polypeptide are fused in-frame to each other.The other polypeptide may be fused to the N-terminus or C-terminus ofthe human c-Maf polypeptide. For example, in one embodiment, the fusionprotein is a GST-human c-Maf fusion protein in which the human c-Mafsequences are fused to the C-terminus of the GST sequences. In anotherembodiment, the fusion protein is a human c-Maf-HA fusion protein inwhich the human c-Maf nucleotide sequence is inserted in a vector suchas pCEP4-HA vector (Herrscher, R. F. et al. (1995) Genes Dev.9:3067-3082) such that the human c-Maf sequences are fused in frame toan influenza hemagglutinin epitope tag. Such fusion proteins canfacilitate the purification of recombinant human c-Maf.

Preferably, a human c-Maf fusion protein of the invention is produced bystandard recombinant DNA techniques. For example, DNA fragments codingfor the different polypeptide sequences are ligated together in-frame inaccordance with conventional techniques, for example employingblunt-ended or stagger-ended termini for ligation, restriction enzymedigestion to provide for appropriate termini, filling-in of cohesiveends as appropriate, alkaline phosphatase treatment to avoid undesirablejoining, and enzymatic ligation. In another embodiment, the fusion genecan be synthesized by conventional techniques including automated DNAsynthesizers. Alternatively, PCR amplification of gene fragments can becarried out using anchor primers which give rise to complementaryoverhangs between two consecutive gene fragments which can subsequentlybe annealed and reamplified to generate a chimeric gene sequence (see,for example, Current Protocols in Molecular Biology, eds. Ausubel et al.John Wiley & Sons: 1992). Moreover, many expression vectors arecommercially available that already encode a fusion moiety (e.g., a GSTpolypeptide or an HA epitope tag). A human c-Maf-encoding nucleic acidcan be cloned into such an expression vector such that the fusion moietyis linked in-frame to the human c-Maf protein.

An isolated human c-Maf protein, or fragment thereof, can be used as animmunogen to generate antibodies that bind specifically to human c-Mafusing standard techniques for polyclonal and monoclonal antibodypreparation. The human c-Maf protein can be used to generate antibodiesor, alternatively, an antigenic peptide fragment of human c-Maf can beused as the immunogen. An antigenic peptide fragment of human c-Maftypically comprises at least 8 amino acid residues of the amino acidsequence shown in SEQ ID NO: 2 and encompasses an epitope of human c-Mafsuch that an antibody raised against the peptide forms a specific immunecomplex with human c-Maf. Preferably, the antigenic peptide comprises atleast 10 amino acid residues, more preferably at least 15 amino acidresidues, even more preferably at least 20 amino acid residues, and mostpreferably at least 30 amino acid residues. Preferred epitopesencompassed by the antigenic peptide are regions of human c-Maf that arelocated on the surface of the protein, e.g., hydrophilic regions, andthat are unique to human c-Maf, as compared to c-Maf proteins from otherspecies, such as chicken or mouse (i.e., an antigenic peptide that spansa region of human c-Maf that is not conserved across species is used asimmunogen; such non-conserved regions/residues are boxed in FIG. 2). Astandard hydrophobicity analysis of the human c-Maf protein can beperformed to identify hydrophilic regions.

A human c-Maf immunogen typically is used to prepare antibodies byimmunizing a suitable subject, (e.g., rabbit, goat, mouse or othermammal) with the immunogen. An appropriate immunogenic preparation cancontain, for examples, recombinantly expressed human c-Maf protein or achemically synthesized human c-Maf peptide. The preparation can furtherinclude an adjuvant, such as Freund's complete or incomplete adjuvant,or similar immunostimulatory agent. Immunization of a suitable subjectwith an immunogenic human c-Maf preparation induces a polyclonalanti-human c-Maf antibody response.

Accordingly, another aspect of the invention pertains to anti-humanc-Maf antibodies. Polyclonal anti-human c-Maf antibodies can be preparedas described above by immunizing a suitable subject with a human c-Mafimmunogen. The anti-human c-Maf antibody titer in the immunized subjectcan be monitored over time by standard techniques, such as with anenzyme linked immunosorbent assay (ELISA) using immobilized human c-Maf.If desired, the antibody molecules directed against human c-Maf can beisolated from the mammal (e.g., from the blood) and further purified bywell known techniques, such as protein A chromatography to obtain theIgG fraction. At an appropriate time after immunization, e.g., when theanti-human c-Maf antibody titers are highest, antibody-producing cellscan be obtained from the subject and used to prepare monoclonalantibodies by standard techniques, such as the hybridoma techniqueoriginally described by Kohler and Milstein (1975, Nature 256:495-497)(see also, Brown et al (1981) J. Immunol 127:539-46; Brown et al. (1980)J Biol Chem 255:4980-83; Yeh et al. (1976) PNAS 76:2927-31; and Yeh etal. (1982) Int. J. Cancer 29:269-75), the more recent human B cellhybridoma technique (Kozbor et al. (1983) Immunol Today 4:72), theEBV-hybridoma technique (Cole et al. (1985), Monoclonal Antibodies andCancer Therapy, Alan R. Liss, Inc., pp. 77-96) or trioma techniques. Thetechnology for producing monoclonal antibody hybridomas is well known(see generally R. H. Kenneth, in Monoclonal Antibodies: A New DimensionIn Biological Analyses, Plenum Publishing Corp., New York, N.Y. (1980);E. A. Lerner (1981) Yale J. Biol. Med., 54:387-402; M. L. Gefter et al.(1977) Somatic Cell Genet., 3:231-36). Briefly, an immortal cell line(typically a myeloma) is fused to lymphocytes (typically splenocytes)from a mammal immunized with a human c-Maf immunogen as described above,and the culture supernatants of the resulting hybridoma cells arescreened to identify a hybridoma producing a monoclonal antibody thatbinds specifically to human c-Maf.

Any of the many well known protocols used for fusing lymphocytes andimmortalized cell lines can be applied for the purpose of generating ananti-human c-Maf monoclonal antibody (see, e.g., G. Galfre et al. (1977)Nature 266:55052; Gefter et al. Somatic Cell Genet., cited supra;Lerner, Yale J. Biol. Med., cited supra; Kenneth, Monoclonal Antibodies,cited supra). Moreover, the ordinary skilled worker will appreciate thatthere are many variations of such methods which also would be useful.Typically, the immortal cell line (e.g., a myeloma cell line) is derivedfrom the same mammalian species as the lymphocytes. For example, murinehybridomas can be made by fusing lymphocytes from a mouse immunized withan immunogenic preparation of the present invention with an immortalizedmouse cell line. Preferred immortal cell lines are mouse myeloma celllines that are sensitive to culture medium containing hypoxanthine,aminopterin and thymidine (“HAT medium”). Any of a number of myelomacell lines may be used as a fusion partner according to standardtechniques, e.g., the P3-NS1/1-Ag4-1, P3-x63-Ag8.653 or Sp2/O-Ag14myeloma lines. These myeloma lines are available from the American TypeCulture Collection (ATCC), Rockville, Md. Typically, HAT-sensitive mousemyeloma cells are fused to mouse splenocytes using polyethylene glycol(“PEG”). Hybridoma cells resulting from the fusion are then selectedusing HAT medium, which kills unfused and unproductively fused myelomacells (unfused splenocytes die after several days because they are nottransformed). Hybridoma cells producing a monoclonal antibody of theinvention are detected by screening the hybridoma culture supernatantsfor antibodies that bind human c-Maf, e.g., using a standard ELISAassay.

Alternative to preparing monoclonal antibody-secreting hybridomas, amonoclonal anti-human c-Maf antibody can be identified and isolated byscreening a recombinant combinatorial immunoglobulin library (e.g., anantibody phage display library) with human c-Maf to thereby isolateimmunoglobulin library members that bind human c-Maf. Kits forgenerating and screening phage display libraries are commerciallyavailable (e.g., the Pharmacia Recombinant Phage Antibody System,Catalog No. 27-9400-01; and the Stratagene SurfZAP™ Phage Display Kit,Catalog No. 240612). Additionally, examples of methods and reagentsparticularly amenable for use in generating and screening antibodydisplay library can be found in, for example, Ladner et al. U.S. Pat.No. 5,223,409; Kang et al. International Publication No. WO 92/18619;Dower et al. International Publication No. WO 91/17271; Winter et al.International Publication WO 92/20791; Markland et al. InternationalPublication No. WO 92/15679; Breitling et al. International PublicationWO 93/01288; McCafferty et al. International Publication No. WO92/01047; Garrard et al. International Publication No. WO 92/09690;Ladner et al. International Publication No. WO 90/02809; Fuchs et al.(1991) Bio/Technology 9:1370-1372; Hay et al. (1992) Hum AntibodHybridomas 3:81-85; Huse et al. (1989) Science 246:1275-1281; Griffithset al. (1993) EMBO J 12:725-734; Hawkins et al (1992) J Mol Biol226:889-896; Clarkson et al. (1991) Nature 352:624-628; Gram et al.(1992) PNAS 89:3576-3580; Garrad et al. (1991) Bio/Technology9:1373-1377; Hoogenboom et al. (1991) Nuc Acid Res 19:4133-4137; Barbaset al. (1991) PNAS 88:7978-7982; and McCafferty et al Nature (1990)348:552-554.

Additionally, recombinant anti-human c-Maf antibodies, such as chimericand humanized monoclonal antibodies, comprising both human and non-humanportions, which can be made using standard recombinant DNA techniques,are within the scope of the invention. Such chimeric and humanizedmonoclonal antibodies can be produced by recombinant DNA techniquesknown in the art, for example using methods described in Robinson et al.International Patent Publication PCT/US86/02269; Akira, et al. EuropeanPatent Application 184,187; Taniguchi, M., European Patent Application171,496; Morrison et al. European Patent Application 173,494; Neubergeret al. PCT Application WO 86/01533; Cabilly et al. U.S. Pat. No.4,816,567; Cabilly et al. European Patent Application 125,023; Better etal. (1988) Science 240:1041-1043; Liu et al. (1987) PNAS 84:3439-3443;Liu et al. (1987) J. Immunol. 139:3521-3526; Sun et al. (1987) PNAS84:214-218; Nishimura et al. (1987) Canc. Res. 47:999-1005; Wood et al.(1985) Nature 314:446-449; and Shaw et al. (1988) J. Natl Cancer Inst.80:1553-1559); Morrison, S. L. (1985) Science 229:1202-1207; Oi et al.(1986) BioTechniques 4:214; Winter U.S. Pat. No. 5,225,539; Jones et al.(1986) Nature 321:552-525; Verhoeyan et al. (1988) Science 239:1534; andBeidler et al. (1988) J. Immunol. 141:4053-4060.

An anti-human c-Maf antibody (e.g., monoclonal antibody) can be used toisolate human c-Maf by standard techniques, such as affinitychromatography or immunoprecipitation. An anti-human c-Maf antibody canfacilitate the purification of natural human c-Maf from cells and ofrecombinantly produced human c-Maf expressed in host cells. Moreover, ananti-human c-Maf antibody can be used to detect human c-Maf protein(e.g., in a cellular lysate or cell supernatant). Detection may befacilitated by coupling (i.e., physically linking) the antibody to adetectable substance. Accordingly, in one embodiment, an anti-humanc-Maf antibody of the invention is labeled with a detectable substance.Examples of detectable substances include various enzymes, prostheticgroups, fluorescent materials, luminescent materials and radioactivematerials. Examples of suitable enzymes include horseradish peroxidase,alkaline phosphatase, β-galactosidase, or acetylcholinesterase; examplesof suitable prosthetic group complexes include streptavidin/biotin andavidin/biotin; examples of suitable fluorescent materials includeumbelliferone, fluorescein, fluorescein isothiocyanate, rhodamine,dichlorotriazinylamine fluorescein, dansyl chloride or phycoerythrin; anexample of a luminescent material includes luminol; and examples ofsuitable radioactive material include ¹²⁵I, ¹³¹I, ³⁵S or ³H.

Yet another aspect of the invention pertains to anti-human c-Mafantibodies that are obtainable by a process comprising:

(a) immunizing an animal with an immunogenic human c-Maf protein, or animmunogenic portion thereof unique to human c-Maf protein; and

(b) isolating from the animal antibodies that specifically bind to ahuman c-Maf protein.

Methods for immunization and recovery of the specific anti-human c-Mafantibodies are described further above.

IV. Pharmaceutical Compositions

Human c-Maf modulators of the invention (e.g., human c-Maf inhibitory orstimulatory agents, including human c-Maf proteins and antibodies) canbe incorporated into pharmaceutical compositions suitable foradministration. Such compositions typically comprise the modulatoryagent and a pharmaceutically acceptable carrier. As used herein the term“pharmaceutically acceptable carrier” is intended to include any and allsolvents, dispersion media, coatings, antibacterial and antifungalagents, isotonic and absorption delaying agents, and the like,compatible with pharmaceutical administration. The use of such media andagents for pharmaceutically active substances is well known in the art.Except insofar as any conventional media or agent is incompatible withthe active compound, use thereof in the compositions is contemplated.Supplementary active compounds can also be incorporated into thecompositions.

A pharmaceutical composition of the invention is formulated to becompatible with its intended route of administration. For example,solutions or suspensions used for parenteral, intradermal, orsubcutaneous application can include the following components: a sterilediluent such as water for injection, saline solution, fixed oils,polyethylene glycols, glycerine, propylene glycol or other syntheticsolvents; antibacterial agents such as benzyl alcohol or methylparabens; antioxidants such as ascorbic acid or sodium bisulfite;chelating agents such as ethylenediaminetetraacetic acid; buffers suchas acetates, citrates or phosphates and agents for the adjustment oftonicity such as sodium chloride or dextrose. pH can be adjusted withacids or bases, such as hydrochloric acid or sodium hydroxide. Theparenteral preparation can be enclosed in ampoules, disposable syringesor multiple dose vials made of glass or plastic.

Pharmaceutical compositions suitable for injectable use include sterileaqueous solutions (where water soluble) or dispersions and sterilepowders for the extemporaneous preparation of sterile injectablesolutions or dispersion. For intravenous administration, suitablecarriers include physiological saline, bacteriostatic water, CremophorEL™ (BASF, Parsippany, N.J.) or phosphate buffered saline (PBS). In allcases, the composition must be sterile and should be fluid to the extentthat easy syringability exists. It must be stable under the conditionsof manufacture and storage and must be preserved against thecontaminating action of microorganisms such as bacteria and fungi. Thecarrier can be a solvent or dispersion medium containing, for example,water, ethanol, polyol (for example, glycerol, propylene glycol, andliquid polyetheylene glycol, and the like), and suitable mixturesthereof. The proper fluidity can be maintained, for example, by the useof a coating such as lecithin, by the maintenance of the requiredparticle size in the case of dispersion and by the use of surfactants.Prevention of the action of microorganisms can be achieved by variousantibacterial and antifungal agents, for example, parabens,chlorobutanol, phenol, ascorbic acid, thimerosal, and the like. In manycases, it will be preferable to include isotonic agents, for example,sugars, polyalcohols such as manitol, sorbitol, sodium chloride in thecomposition. Prolonged absorption of the injectable compositions can bebrought about by including in the composition an agent which delaysabsorption, for example, aluminum monostearate and gelatin.

Sterile injectable solutions can be prepared by incorporating the activecompound in the required amount in an appropriate solvent with one or acombination of ingredients enumerated above, as required, followed byfiltered sterilization. Generally, dispersions are prepared byincorporating the active compound into a sterile vehicle which containsa basic dispersion medium and the required other ingredients from thoseenumerated above. In the case of sterile powders for the preparation ofsterile injectable solutions, the preferred methods of preparation arevacuum drying and freeze-drying which yields a powder of the activeingredient plus any additional desired ingredient from a previouslysterile-filtered solution thereof.

Oral compositions generally include an inert diluent or an ediblecarrier. They can be enclosed in gelatin capsules or compressed intotablets. For the purpose of oral therapeutic administration, the activecompound can be incorporated with excipients and used in the form oftablets, troches, or capsules. Oral compositions can also be preparedusing a fluid carrier for use as a mouthwash, wherein the compound inthe fluid carrier is applied orally and swished and expectorated orswallowed. Pharmaceutically compatible binding agents, and/or adjuvantmaterials can be included as part of the composition. The tablets,pills, capsules, troches and the like can contain any of the followingingredients, or compounds of a similar nature: a binder such asmicrocrystalline cellulose, gum tragacanth or gelatin; an excipient suchas starch or lactose, a disintegrating agent such as alginic acid,Primogel, or corn starch; a lubricant such as magnesium stearate orSterotes; a glidant such as colloidal silicon dioxide; a sweeteningagent such as sucrose or saccharin; or a flavoring agent such aspeppermint, methyl salicylate, or orange flavoring.

In one embodiment, the active compounds are prepared with carriers thatwill protect the compound against rapid elimination from the body, suchas a controlled release formulation, including implants andmicroencapsulated delivery systems. Biodegradable, biocompatiblepolymers can be used, such as ethylene vinyl acetate, polyanhydrides,polyglycolic acid, collagen, polyorthoesters, and polylactic acid.Methods for preparation of such formulations will be apparent to thoseskilled in the art. The materials can also be obtained commercially fromAlza Corporation and Nova Pharmaceuticals, Inc. Liposomal suspensions(including liposomes targeted to infected cells with monoclonalantibodies to viral antigens) can also be used as pharmaceuticallyacceptable carriers. These may be prepared according to methods known tothose skilled in the art, for example, as described in U.S. Pat. No.4,522,811.

V. Methods of the Invention

Another aspect of the invention pertains to methods of using the varioushuman c-Maf compositions of the invention. For example, the inventionprovides a method for detecting the presence of human c-Maf activity ina biological sample. The method involves contacting the biologicalsample with an agent capable of detecting human c-Maf activity, such ashuman c-Maf protein or human c-Maf mRNA, such that the presence of humanc-Maf activity is detected in the biological sample.

A preferred agent for detecting human c-Maf mRNA is a labeled nucleicacid probe capable of specifically hybridizing to human c-Maf mRNA. Thenucleic acid probe can be, for example, the human c-Maf DNA of SEQ IDNO: 1 (or plasmid pHu-c-Maf), or a portion thereof unique to human c-Maf(as compared to c-Maf from other species, such as chicken or mouse),such as an oligonucleotide of at least 15, 30, 50, 100, 200, 300, 400,500, 600, 700, 800, 900 or 1000 nucleotides in length and sufficient tospecifically hybridize under stringent conditions to human c-Maf mRNA.

A preferred agent for detecting human c-Maf protein is a labeledantibody capable of binding to human c-Maf protein. Antibodies can bepolyclonal, or more preferably, monoclonal. An intact antibody, or afragment thereof (e.g., Fab or F(ab′)₂) can be used. The term “labeled”,with regard to the probe or antibody, is intended to encompass directlabeling of the probe or antibody by coupling (i.e., physically linking)a detectable substance to the probe or antibody, as well as indirectlabeling of the probe or antibody by reactivity with another reagentthat is directly labeled. Examples of indirect labeling includedetection of a primary antibody using a fluorescently labeled secondaryantibody and end-labeling of a DNA probe with biotin such that it can bedetected with fluorescently labeled streptavidin. The term “biologicalsample” is intended to include tissues, cells and biological fluids. Forexample, techniques for detection of human c-Maf mRNA include Northernhybridizations and in situ hybridizations. Techniques for detection ofhuman c-Maf protein include enzyme linked immunosorbent assays (ELISAs),Western blots, immunoprecipitations and immunofluorescence.

The invention further provides methods for identifying compounds thatmodulate the activity of a human c-Maf protein. For example, theinvention provides a method for identifying a compound that modulatesthe activity of a human c-Maf protein, comprising

providing an indicator composition that comprises a human c-Maf protein;

contacting the indicator composition with a test compound; and

determining the effect of the test compound on the activity of the humanc-Maf protein in the indicator composition to thereby identify acompound that modulates the activity of a human c-Maf protein.

Specific embodiments of the screening methods of the invention exploitthe ability of c-Maf proteins to bind to DNA (e.g., the ability to bindto a Maf Response Element (MARE)) and/or to regulate gene expression(e.g., regulate expression of a Th2-associated cytokine gene). Forfurther description of these activities of Maf proteins, in general, seefor example PCT Publication WO 97/39721, Kurschner and Morgan (1995)Mol. Cell. Biol., 15:246-254; Kataoka et al. (1993) J. Virol.67:2133-2141; Kataoka et al. (1996) Oncogene 12:53-62; Kataoka et al.(1994) Mol. Cell. Biol. 14:700-712; and Ho, I-C. et al. (1996) Cell85:973-983; the contents of each of which are expressly incorporatedherein by reference.

In a preferred embodiment of the screening assays of the invention, theindicator composition comprises an indicator cell, wherein saidindicator cell comprises: (i) the a human c-Maf protein and (ii) areporter gene responsive to the human c-Maf protein. Preferably, theindicator cell contains:

-   -   i) a recombinant expression vector encoding the human c-Maf; and    -   ii) a vector comprising regulatory sequences of a Th2-associated        cytokine gene operatively linked a reporter gene; and        said method comprises:

a) contacting the indicator cell with a test compound;

b) determining the level of expression of the reporter gene in theindicator cell in the presence of the test compound; and

c) comparing the level of expression of the reporter gene in theindicator cell in the presence of the test compound with the level ofexpression of the reporter gene in the indicator cell in the absence ofthe test compound to thereby identify a compound that modulates theactivity of human c-Maf.

In another preferred embodiment, the indicator composition comprises apreparation of: (i) a human c-Maf protein and (ii) a DNA molecule towhich the human c-Maf binds, and

said method comprises:

a) contacting the indicator composition with a test compound;

b) determining the degree of interaction of the human c-Maf protein andthe DNA molecule in the presence of the test compound; and

c) comparing the degree of interaction of the human c-Maf and the DNAmolecule in the presence of the test compound with the degree ofinteraction of the human c-Maf protein and the DNA molecule in theabsence of the test compound to thereby identify a compound thatmodulates the activity of human c-Maf.

Preferably, the DNA molecule to which human c-Maf binds comprises a mafresponse element (MARE).

In another preferred embodiment, the method identifies proteins thatinteract with human c-Maf. In this embodiment,

the indicator composition is an indicator cell, which indicator cellcomprises:

-   -   i) a reporter gene operably linked to a transcriptional        regulatory sequence; and    -   ii) a first chimeric gene which encodes a first fusion protein,        said first fusion protein including human c-Maf;

the test compound comprises a library of second chimeric genes, whichlibrary encodes second fusion proteins;

expression of the reporter gene being sensitive to interactions betweenthe first fusion protein, the second fusion protein and thetranscriptional regulatory sequence; and

wherein the effect of the test compound on human c-Maf in the indicatorcomposition is determined by determining the level of expression of thereporter gene in the indicator cell to thereby identify a test compoundcomprising a protein that interacts with human c-Maf.

In a preferred embodiment, the library of second chimeric genes isprepared from cDNA library from Th2 cells.

In a preferred embodiment of the screening assays of the invention, oncea test compound is identified as modulating the activity of human c-Maf,the effect of the test compound on an immune response is then tested.Accordingly, the screening methods of the invention can further comprisedetermining the effect of the compound on an immune response to therebyidentify a compound that modulates an immune response. In oneembodiment, the effect of the compound on an immune response isdetermined by determining the effect of the compound on expression of aTh2-associated cytokine gene, such as an interleukin-4 gene. As usedherein, the term “Th2-associated cytokine” is intended to refer to acytokine that is produced preferentially or exclusively by Th2 cellsrather than by Th1 cells. Examples of Th2-associated cytokines includeIL-4, IL-5, IL-6 and IL-13. In another embodiment, the effect of thecompound of interest on an immune response is determined by determiningthe effect of the compound on development of T helper type 1 (Th1) or Thelper type 2 (Th2) cells.

Recombinant expression vectors that can be used for expression of humanc-Maf in the indicator cell are known in the art (see discussionsabove). In one embodiment, within the expression vector the humanc-Maf-coding sequences are operatively linked to regulatory sequencesthat allow for constitutive expression of human c-Maf in the indicatorcell (e.g., viral regulatory sequences, such as a cytomegaloviruspromoter/enhancer, can be used). Use of a recombinant expression vectorthat allows for constitutive expression of human c-Maf in the indicatorcell is preferred for identification of compounds that enhance orinhibit the activity of human c-Maf. In an alternative embodiment,within the expression vector the human c-Maf-coding sequences areoperatively linked to regulatory sequences of the endogenous human c-Mafgene (i.e., the promoter regulatory region derived from the endogenoushuman c-Maf gene). Use of a recombinant expression vector in which humanc-Maf expression is controlled by the endogenous regulatory sequences ispreferred for identification of compounds that enhance or inhibit thetranscriptional expression of human c-Maf.

In methods in which a Th2-associated cytokine gene is utilized (e.g., asa reporter gene), preferably, the Th2-associated cytokine isinterleukin-4. It has previously shown that Th2-specific, inducible IL-4expression can be directed by as little as 157 bp of the proximal IL-4promoter in Th2 cells (Hodge, M. et al. (1995) J. Immunol.154:6397-6405). Accordingly, in one embodiment, a method of theinvention utilizes a reporter gene construct containing this region ofthe proximal IL-4 promoter, most preferably nucleotides −157 to +58(relative to the start site of transcription at +1) of the IL-4promoter. Alternatively, stronger reporter gene expression can beachieved using a longer portion of the IL-4 upstream regulatory region,such as about 3 kb of upstream regulatory sequences. Suitable reportergene constructs are described in Todd, M. et al. (1993) J. Exp. Med.177:1663-1674. See also PCT Publication WO 97/39721.

A variety of reporter genes are known in the art and are suitable foruse in the screening assays of the invention. Examples of suitablereporter genes include those which encode chloramphenicolacetyltransferase, beta-galactosidase, alkaline phosphatase orluciferase. Standard methods for measuring the activity of these geneproducts are known in the art.

A variety of cell types are suitable for use as an indicator cell in thescreening assay. Preferably a cell line is used which does not normallyexpress human c-Maf, such as a B cell (e.g., the M12 B lymphoma cellline) or a Th1 cell clone (e.g., AE7 cells). Nonlymphoid cell lines canalso be used as indicator cells, such as the HepG2 hepatoma cell line.Yeast cells also can be used as indicator cells.

In one embodiment, the level of expression of the reporter gene in theindicator cell in the presence of the test compound is higher than thelevel of expression of the reporter gene in the indicator cell in theabsence of the test compound and the test compound is identified as acompound that stimulates the expression or activity of human c-Maf. Inanother embodiment, the level of expression of the reporter gene in theindicator cell in the presence of the test compound is lower than thelevel of expression of the reporter gene in the indicator cell in theabsence of the test compound and the test compound is identified as acompound that inhibits the expression or activity of human c-Maf.

Alternative to the use of a reporter gene construct, compounds thatmodulate the expression or activity of human c-Maf can be identified byusing other “read-outs.” For example, an indicator cell can betransfected with a human c-Maf expression vector, incubated in thepresence and in the absence of a test compound, and Th2-associatedcytokine production can be assessed by detecting cytokine mRNA (e.g.,IL-4 mRNA) in the indicator cell or cytokine secretion (i.e., IL-4secretion) into the culture supernatant. Standard methods for detectingcytokine mRNA, such as reverse transcription-polymerase chain reaction(RT-PCR) are known in the art. Standard methods for detecting cytokineprotein in culture supernatants, such as enzyme linked immunosorbentassays (ELISA) are also known in the art.

As described above, the invention provides a screening assay foridentifying proteins (e.g., proteins in Th2 cells) that interact withhuman c-Maf. These assays can be designed based on the two-hybrid assaysystem (also referred to as an interaction trap assay) known in the art(see e.g., Field U.S. Pat. No. 5,283,173; Zervos et al. (1993) Cell72:223-232; Madura et al. (1993) J. Biol. Chem. 268:12046-12054; Bartelet al. (1993) Biotechniques 14:920-924; and Iwabuchi et al. (1993)Oncogene 8:1693-1696). The two-hybrid assay is generally used foridentifying proteins that interact with a particular target protein. Theassay employs gene fusions to identify proteins capable of interactingto reconstitute a functional transcriptional activator. Thetranscriptional activator consists of a DNA-binding domain and atranscriptional activation domain, wherein both domains are required toactivate transcription of genes downstream from a target sequence (suchas an upstream activator sequence (UAS) for GAL4). DNA sequencesencoding a target “bait” protein are fused to either of these domainsand a library of DNA sequences is fused to the other domain. “Fish”fusion proteins (generated from the fusion library) capable of bindingto the target-fusion protein (e.g., a target GAL4-fusion “bait”) willgenerally bring the two domains (DNA-binding domain and transcriptionalactivation domain) into close enough proximity to activate thetranscription of a reporter gene inserted downstream from the targetsequence. Thus, the “fish” proteins can be identified by their abilityto reconstitute a functional transcriptional activator (e.g., afunctional GAL4 transactivator).

This general two-hybrid system can be applied to the identification ofproteins in cells (e.g., Th2 cells) that interact with human c-Maf byconstruction of a target human c-Maf fusion protein (e.g., a humanc-Maf/GAL4 binding domain fusion as the “bait”) and a cDNA library of“fish” fusion proteins (e.g., a cDNA/GAL4 activation domain library),wherein the cDNA library is prepared from mRNA of a cell type ofinterest (e.g., Th2 cells), and introducing these constructs into a hostcell that also contains a reporter gene construct linked to a regulatorysequence responsive to human c-Maf (e.g., a MARE sequence, for example aregion of the IL-4 promoter, as discussed above). cDNAs encodingproteins that interact with human c-Maf can be identified based upontransactivation of the reporter gene construct.

Alternatively, a “single-hybrid” assay, such as that described inSieweke, M. H. et al. (1996) Cell 85:49-60, can be used to identifyproteins that interact with human c-Maf. This assay is a modification ofthe two-hybrid system discussed above. In this system, the “bait” is atranscription factor from which the transactivation domain has beenremoved (e.g., human c-Maf from which the amino-terminal transactivationdomain has been removed) and the “fish” is a non-fusion cDNA library(e.g., a cDNA library prepared from Th2 cells). These constructs areintroduced into host cells (e.g., yeast cells) that also contains areporter gene construct linked to a regulatory sequence responsive tohuman c-Maf (e.g., a MARE sequence, for example a region of the IL-4promoter, responsive to human c-Maf). cDNAs encoding proteins thatinteract with human c-Maf can be identified based upon transactivationof the reporter gene construct.

As described above, the invention provides a screening assay foridentifying compounds that modulate the interaction of human c-Maf and aMARE (e.g., a MARE in an IL-4 gene regulatory region). Assays are knownin the art that detect the interaction of a DNA binding protein with atarget DNA sequence (e.g., electrophoretic mobility shift assays, DNAseI footprinting assays and the like). By performing such assays in thepresence and absence of test compounds, these assays can be used toidentify compounds that modulate (e.g., inhibit or enhance) theinteraction of the DNA binding protein with its target DNA sequence.

In one embodiment, the amount of binding of human c-Maf to the DNAfragment in the presence of the test compound is greater than the amountof binding of human c-Maf to the DNA fragment in the absence of the testcompound, in which case the test compound is identified as a compoundthat enhances binding of human c-Maf. In another embodiment, the amountof binding of human c-Maf to the DNA fragment in the presence of thetest compound is less than the amount of binding of human c-Maf to theDNA fragment in the absence of the test compound, in which case the testcompound is identified as a compound that inhibits binding of humanc-Maf.

Yet another aspect of the invention pertains to methods of modulatinghuman c-Maf activity in a cell. The modulatory methods of the inventioninvolve contacting the cell with an agent that modulates human c-Mafactivity such that human c-Maf activity in the cell is modulated. Theagent may act by modulating the activity of human c-Maf protein in thecell or by modulating transcription of the human c-Maf gene ortranslation of the human c-Maf mRNA. As used herein, the term“modulating” is intended to include inhibiting or decreasing human c-Mafactivity and stimulating or increasing human c-Maf activity.Accordingly, in one embodiment, the agent inhibits human c-Maf activity.In another embodiment, the agent stimulates human c-Maf activity.

A. Inhibitory Agents

According to a modulatory method of the invention, human c-Maf activityis inhibited in a cell by contacting the cell with an inhibitory agent.Inhibitory agents of the invention can be, for example, intracellularbinding molecules that act to inhibit the expression or activity ofhuman c-Maf. As used herein, the term “intracellular binding molecule”is intended to include molecules that act intracellularly to inhibit theexpression or activity of a protein by binding to the protein itself, toa nucleic acid (e.g., an mRNA molecule) that encodes the protein or to atarget with which the protein normally interacts (e.g., to a DNA targetsequence to which c-Maf binds). Examples of intracellular bindingmolecules, described in further detail below, include antisense humanc-Maf nucleic acid molecules (e.g., to inhibit translation of humanc-Maf mRNA), intracellular anti-human c-Maf antibodies (e.g., to inhibitthe activity of human c-Maf protein) and dominant negative mutants ofthe human c-Maf protein.

In one embodiment, an inhibitory agent of the invention is an antisensenucleic acid molecule that is complementary to a gene encoding humanc-Maf or to a portion of said gene, or a recombinant expression vectorencoding said antisense nucleic acid molecule. The use of antisensenucleic acids to downregulate the expression of a particular protein ina cell is well known in the art (see e.g., Weintraub, H. et al.,Antisense RNA as a molecular tool for genetic analysis, Reviews—Trendsin Genetics, Vol. 1(1) 1986; Askari, F. K. and McDonnell, W. M. (1996)N. Eng. J. Med. 334:316-318; Bennett, M. R. and Schwartz, S. M. (1995)Circulation 92:1981-1993; Mercola, D. and Cohen, J. S. (1995) CancerGene Ther. 2:47-59; Rossi, J. J. (1995) Br. Med. Bull. 51:217-225;Wagner, R. W. (1994) Nature 372:333-335). An antisense nucleic acidmolecule comprises a nucleotide sequence that is complementary to thecoding strand of another nucleic acid molecule (e.g., an mRNA sequence)and accordingly is capable of hydrogen bonding to the coding strand ofthe other nucleic acid molecule. Antisense sequences complementary to asequence of an mRNA can be complementary to a sequence found in thecoding region of the mRNA, the 5′ or 3′ untranslated region of the mRNAor a region bridging the coding region and an untranslated region (e.g.,at the junction of the 5′ untranslated region and the coding region).Furthermore, an antisense nucleic acid can be complementary in sequenceto a regulatory region of the gene encoding the mRNA, for instance atranscription initiation sequence or regulatory element. Preferably, anantisense nucleic acid is designed so as to be complementary to a regionpreceding or spanning the initiation codon on the coding strand or inthe 3′ untranslated region of an mRNA. An antisense nucleic acid forinhibiting the expression of human c-Maf protein in a cell can bedesigned based upon the nucleotide sequence encoding the human c-Mafprotein (e.g., SEQ ID NO: 1 and plasmid pHu-c-Maf), constructedaccording to the rules of Watson and Crick base pairing.

An antisense nucleic acid can exist in a variety of different forms. Forexample, the antisense nucleic acid can be an oligonucleotide that iscomplementary to only a portion of a human c-Maf gene. An antisenseoligonucleotides can be constructed using chemical synthesis proceduresknown in the art. An antisense oligonucleotide can be chemicallysynthesized using naturally occurring nucleotides or variously modifiednucleotides designed to increase the biological stability of themolecules or to increase the physical stability of the duplex formedbetween the antisense and sense nucleic acids, e.g. phosphorothioatederivatives and acridine substituted nucleotides can be used. To inhibithuman c-Maf expression in cells in culture, one or more antisenseoligonucleotides can be added to cells in culture media, typically atabout 200 μg oligonucleotide/ml.

Alternatively, an antisense nucleic acid can be produced biologicallyusing an expression vector into which a nucleic acid has been subclonedin an antisense orientation (i e. nucleic acid transcribed from theinserted nucleic acid will be of an antisense orientation to a targetnucleic acid of interest). Regulatory sequences operatively linked to anucleic acid cloned in the antisense orientation can be chosen whichdirect the expression of the antisense RNA molecule in a cell ofinterest, for instance promoters and/or enhancers or other regulatorysequences can be chosen which direct constitutive, tissue specific orinducible expression of antisense RNA. For example, for inducibleexpression of antisense RNA, an inducible eukaryotic regulatory system,such as the Tet system (e.g., as described in Gossen, M. and Bujard, H.(1992) Proc. Natl. Acad. Sci. USA 89:5547-5551; Gossen, M. et al. (1995)Science 268:1766-1769; PCT Publication No. WO 94/29442; and PCTPublication No. WO 96/01313) can be used. The antisense expressionvector is prepared as described above for recombinant expressionvectors, except that the cDNA (or portion thereof) is cloned into thevector in the antisense orientation. The antisense expression vector canbe in the form of, for example, a recombinant plasmid, phagemid orattenuated virus. The antisense expression vector is introduced intocells using a standard transfection technique, as described above forrecombinant expression vectors.

In another embodiment, an antisense nucleic acid for use as aninhibitory agent is a ribozyme. Ribozymes are catalytic RNA moleculeswith ribonuclease activity which are capable of cleaving asingle-stranded nucleic acid, such as an mRNA, to which they have acomplementary region (for reviews on ribozymes see e.g., Ohkawa, J. etal. (1995) J. Biochem. 118:251-258; Sigurdsson, S. T. and Eckstein, F.(1995) Trends Biotechnol. 13:286-289; Rossi, J. J. (1995) TrendsBiotechnol. 13:301-306; Kiehntopf, M. et al. (1995) J. Mol. Med.73:65-71). A ribozyme having specificity for human c-Maf mRNA can bedesigned based upon the nucleotide sequence of the human c-Maf cDNA. Forexample, a derivative of a Tetrahymena L-19 IVS RNA can be constructedin which the base sequence of the active site is complementary to thebase sequence to be cleaved in a human c-Maf mRNA. See for example U.S.Pat. Nos. 4,987,071 and 5,116,742, both by Cech et al. Alternatively,human c-Maf mRNA can be used to select a catalytic RNA having a specificribonuclease activity from a pool of RNA molecules. See for exampleBartel, D. and Szostak, J. W. (1993) Science 261: 1411-1418.

Another type of inhibitory agent that can be used to inhibit theexpression and/or activity of human c-Maf in a cell is an intracellularantibody specific for the human c-Maf protein. The use of intracellularantibodies to inhibit protein function in a cell is known in the art(see e.g., Carlson, J. R. (1988) Mol. Cell. Biol. 8:2638-2646; Biocca,S. et al. (1990) EMBO J. 9:101-108; Werge, T. M. et al. (1990) FEBSLetters 274:193-198; Carlson, J. R. (1993) Proc. Natl. Acad. Sci. USA90:7427-7428; Marasco, W. A. et al. (1993) Proc. Natl. Acad. Sci. USA90:7889-7893; Biocca, S. et al. (1994) Bio/Technology 12:396-399; Chen,S-Y. et al. (1994) Human Gene Therapy 5:595-601; Duan, L et al. (1994)Proc. Natl. Acad. Sci. USA 91:5075-5079; Chen, S-Y. et al. (1994) Proc.Natl. Acad. Sci. USA 91:5932-5936; Beerli, R. R. et al. (1994) J. Biol.Chem. 269:23931-23936; Beerli, R. R. et al. (1994) Biochem. Biophys.Res. Commun. 204:666-672; Mhashilkar, A. M. et al. (1995) EMBO J.14:1542-1551; Richardson, J. H. et al. (1995) Proc. Natl. Acad. Sci. USA92:3137-3141; PCT Publication No. WO 94/02610 by Marasco et al.; and PCTPublication No. WO 95/03832 by Duan et al.).

To inhibit protein activity using an intracellular antibody, arecombinant expression vector is prepared which encodes the antibodychains in a form such that, upon introduction of the vector into a cell,the antibody chains are expressed as a functional antibody in anintracellular compartment of the cell. For inhibition of human c-Mafactivity according to the inhibitory methods of the invention, anintracellular antibody that specifically binds the human c-Maf proteinis expressed in the cytoplasm of the cell. To prepare an intracellularantibody expression vector, antibody light and heavy chain cDNAsencoding antibody chains specific for the target protein of interest,e.g., human c-Maf, are isolated, typically from a hybridoma thatsecretes a monoclonal antibody specific for the human c-Maf protein.Hybridomas secreting anti-human c-Maf monoclonal antibodies, orrecombinant anti-human c-Maf monoclonal antibodies, can be prepared asdescribed above. Once a monoclonal antibody specific for human c-Mafprotein has been identified (e.g., either a hybridoma-derived monoclonalantibody or a recombinant antibody from a combinatorial library), DNAsencoding the light and heavy chains of the monoclonal antibody areisolated by standard molecular biology techniques. For hybridoma derivedantibodies, light and heavy chain cDNAs can be obtained, for example, byPCR amplification or cDNA library screening. For recombinant antibodies,such as from a phage display library, cDNA encoding the light and heavychains can be recovered from the display package (e.g., phage) isolatedduring the library screening process. Nucleotide sequences of antibodylight and heavy chain genes from which PCR primers or cDNA libraryprobes can be prepared are known in the art. For example, many suchsequences are disclosed in Kabat, E. A., et al. (1991) Sequences ofProteins of Immunological Interest, Fifth Edition, U.S. Department ofHealth and Human Services, NIH Publication No. 91-3242 and in the“Vbase” human germline sequence database.

Once obtained, the antibody light and heavy chain sequences are clonedinto a recombinant expression vector using standard methods. To allowfor cytoplasmic expression of the light and heavy chains, the nucleotidesequences encoding the hydrophobic leaders of the light and heavy chainsare removed. An intracellular antibody expression vector can encode anintracellular antibody in one of several different forms. For example,in one embodiment, the vector encodes full-length antibody light andheavy chains such that a full-length antibody is expressedintracellularly. In another embodiment, the vector encodes a full lengthlight chain but only the VH/CH1 region of the heavy chain such that aFab fragment is expressed intracellularly. In the most preferredembodiment, the vector encodes a single chain antibody (scFv) whereinthe variable regions of the light and heavy chains are linked by aflexible peptide linker (e.g., (Gly₄Ser)₃) and expressed as a singlechain molecule. To inhibit human c-Maf activity in a cell, theexpression vector encoding the anti-human c-Maf intracellular antibodyis introduced into the cell by standard transfection methods, asdiscussed hereinbefore.

Yet another form of an inhibitory agent of the invention is aninhibitory form of human c-Maf, also referred to herein as a dominantnegative inhibitor. The maf family of proteins are known to homodimerizeand to heterodimerize with other AP-1 family members, such as Fos andJun (see e.g., Kerppola, T. K. and Curran, T. (1994) Oncogene 9:675-684;Kataoka, K. et al. (1994) Mol. Cell. Biol. 14:700-712). One means toinhibit the activity of transcription factors that form dimers isthrough the use of a dominant negative inhibitor that has the ability todimerize with functional transcription factors but that lacks theability to activate transcription (see e.g., Petrak, D. et al. (1994) J.Immunol. 153:2046-2051). By dimerizing with functional transcriptionfactors, such dominant negative inhibitors can inhibit their functionalactivity. This process may occur naturally as a means to regulate geneexpression. For example, there are a number of “small” maf proteins,such as mafK, mafF, mafG and p18, which lack the amino terminal twothirds of c-Maf that contains the transactivating domain (Fujiwara, K.T. et al. (1993) Oncogene 8:2371-2380; Igarashi, K. et al. (1995) J.Biol. Chem. 270:7615-7624; Andrews, N. C. et al. (1993) Proc. Natl.Acad. Sci. USA 90:11488-11492; Kataoka, K. et al. (1995) Mol. Cell.Biol. 15:2180-2190). Homodimers of the small maf proteins act asnegative regulators of transcription (Igarashi, K. et al. (1994) Nature367:568-572) and three of the small maf proteins (MafK, MafF and MafG)have been shown to competitively inhibit transactivation mediated by thev-Maf oncoprotein (Kataoka, K. et al. (1996) Oncogene 12:53-62).Additionally, MafB has been identified as an interaction partner ofEts-1 and shown to inhibit Ets-1-mediated transactivation of thetransferrin receptor and to inhibit erythroid differentiation (Sieweke,M. H. et al. (1996) Cell 85:49-60).

Accordingly, an inhibitory agent of the invention can be a form of ahuman c-Maf protein that has the ability to dimerize with other proteinsbut that lacks the ability to activate transcription. This dominantnegative form of a human c-Maf protein may be, for example, a mutatedform of human c-Maf in which the transactivation domain has beenremoved. Such dominant negative human c-Maf proteins can be expressed incells using a recombinant expression vector encoding the human c-Mafprotein, which is introduced into the cell by standard transfectionmethods. To express a mutant form of human c-Maf lacking atransactivation domain, nucleotide sequences encoding the amino terminaltransactivation domain of human c-Maf are removed from the c-maf codingsequences by standard recombinant DNA techniques. Preferably, at leastamino acids 1-122 are removed. More preferably, at least amino acids1-187, or amino acids 1-257, are removed. Nucleotide sequences encodingthe basic-leucine zipper region are maintained. The truncated DNA isinserted into a recombinant expression vector, which is then introducedinto a cell to allow for expression of the truncated human c-Maf,lacking a transactivation domain, in the cell.

Other inhibitory agents that can be used to inhibit the activity of ahuman c-Maf protein are chemical compounds that directly inhibit humanc-Maf activity or inhibit the interaction between human c-Maf and targetDNA or another protein. Such compounds can be identified using screeningassays that select for such compounds, as described in detail above.

B. Stimulatory Agents

According to a modulatory method of the invention, human c-Maf activityis stimulated in a cell by contacting the cell with a stimulatory agent.Examples of such stimulatory agents include active human c-Maf proteinand nucleic acid molecules encoding human c-Maf that are introduced intothe cell to increase human c-Maf activity in the cell. A preferredstimulatory agent is a nucleic acid molecule encoding a human c-Mafprotein, wherein the nucleic acid molecule is introduced into the cellin a form suitable for expression of the active human c-Maf protein inthe cell. To express a human c-Maf protein in a cell, typically a humanc-Maf-encoding DNA is first introduced into a recombinant expressionvector using standard molecular biology techniques, as described herein.A human c-Maf-encoding DNA can be obtained, for example, from plasmidpHu-c-Maf or by amplification using the polymerase chain reaction (PCR),using primers based on the human c-Maf nucleotide sequence. Followingisolation or amplification of human c-Maf-encoding DNA, the DNA fragmentis introduced into an expression vector and transfected into targetcells by standard methods, as described herein.

Other stimulatory agents that can be used to stimulate the activity of ahuman c-Maf protein are chemical compounds that stimulate human c-Mafactivity in cells, such as compounds that directly stimulate human c-Mafprotein and compounds that promote the interaction between human c-Mafand target DNA or other proteins. Such compounds can be identified usingscreening assays that select for such compounds, as described in detailabove.

The modulatory methods of the invention can be performed in vitro (e.g.,by culturing the cell with the agent or by introducing the agent intocells in culture) or, alternatively, in vivo (e.g., by administering theagent to a subject or by introducing the agent into cells of a subject,such as by gene therapy). For practicing the modulatory method in vitro,cells can be obtained from a subject by standard methods and incubated(i.e., cultured) in vitro with a modulatory agent of the invention tomodulate human c-Maf activity in the cells. For example, peripheralblood mononuclear cells (PBMCs) can be obtained from a subject andisolated by density gradient centrifugation, e.g., with Ficoll/Hypaque.Specific cell populations can be depleted or enriched using standardmethods. For example, monocytes/macrophages can be isolated by adherenceon plastic. B cells can be enriched for example, by positive selectionusing antibodies to B cell surface markers, for example by incubatingcells with a specific primary monoclonal antibody (mAb), followed byisolation of cells that bind the mAb using magnetic beads coated with asecondary antibody that binds the primary mAb. Specific cell populationscan also be isolated by fluorescence activated cell sorting according tostandard methods. If desired, cells treated in vitro with a modulatoryagent of the invention can be readministered to the subject. Foradministration to a subject, it may be preferable to first removeresidual agents in the culture from the cells before administering themto the subject. This can be done for example by a Ficoll/Hypaquegradient centrifugation of the cells. For further discussion of ex vivogenetic modification of cells followed by readministration to a subject,see also U.S. Pat. No. 5,399,346 by W. F. Anderson et al.

For practicing the modulatory method in vivo in a subject, themodulatory agent can be administered to the subject such that humanc-Maf activity in cells of the subject is modulated. The term “subject”is intended to include living organisms in which an immune response canbe elicited. Preferred subjects are mammals. Examples of subjectsinclude humans, monkeys, dogs, cats, mice, rats, cows, horses, goats andsheep.

For stimulatory or inhibitory agents that comprise nucleic acids(including recombinant expression vectors encoding human c-Maf protein,antisense RNA, intracellular antibodies or dominant negativeinhibitors), the agents can be introduced into cells of the subjectusing methods known in the art for introducing nucleic acid (e.g., DNA)into cells in vivo. Examples of such methods encompass both non-viraland viral methods, including:

Direct Injection:

Naked DNA can be introduced into cells in vivo by directly injecting theDNA into the cells (see e.g., Acsadi et al. (1991) Nature 332:815-818;Wolff et al. (1990) Science 247:1465-1468). For example, a deliveryapparatus (e.g., a “gene gun”) for injecting DNA into cells in vivo canbe used. Such an apparatus is commercially available (e.g., fromBioRad).

Cationic Lipids:

Naked DNA can be introduced into cells in vivo by complexing the DNAwith cationic lipids or encapsulating the DNA in cationic liposomes.Examples of suitable cationic lipid formulations includeN-[-1-(2,3-dioleoyloxy)propyl]N,N,N-triethylammonium chloride (DOTMA)and a 1:1 molar ratio of1,2-dimyristyloxy-propyl-3-dimethylhydroxyethylammonium bromide (DMRIE)and dioleoyl phosphatidylethanolamine (DOPE) (see e.g., Logan, J. J. etal. (1995) Gene Therapy 2:38-49; San, H. et al. (1993) Human GeneTherapy 4:781-788).

Receptor-Mediated DNA Uptake:

Naked DNA can also be introduced into cells in vivo by complexing theDNA to a cation, such as polylysine, which is coupled to a ligand for acell-surface receptor (see for example Wu, G. and Wu, C. H. (1988) J.Biol. Chem. 263:14621; Wilson et al. (1992) J. Biol. Chem. 267:963-967;and U.S. Pat. No. 5,166,320). Binding of the DNA-ligand complex to thereceptor facilitates uptake of the DNA by receptor-mediated endocytosis.A DNA-ligand complex linked to adenovirus capsids which naturallydisrupt endosomes, thereby releasing material into the cytoplasm can beused to avoid degradation of the complex by intracellular lysosomes (seefor example Curiel et al. (1991) Proc. Natl. Acad. Sci. USA 88:8850;Cristiano et al. (1993) Proc. Natl. Acad. Sci. USA 90:2122-2126).

Retroviruses:

Defective retroviruses are well characterized for use in gene transferfor gene therapy purposes (for a review see Miller, A. D. (1990) Blood76:271). A recombinant retrovirus can be constructed having a nucleotidesequences of interest incorporated into the retroviral genome.Additionally, portions of the retroviral genome can be removed to renderthe retrovirus replication defective. The replication defectiveretrovirus is then packaged into virions which can be used to infect atarget cell through the use of a helper virus by standard techniques.Protocols for producing recombinant retroviruses and for infecting cellsin vitro or in vivo with such viruses can be found in Current Protocolsin Molecular Biology, Ausubel, F. M. et al. (eds.) Greene PublishingAssociates, (1989), Sections 9.10-9.14 and other standard laboratorymanuals. Examples of suitable retroviruses include pLJ, pZIP, pWE andpEM which are well known to those skilled in the art. Examples ofsuitable packaging virus lines include ψ Crip, ψCre, ψ2 and ψAm.Retroviruses have been used to introduce a variety of genes into manydifferent cell types, including epithelial cells, endothelial cells,lymphocytes, myoblasts, hepatocytes, bone marrow cells, in vitro and/orin vivo (see for example Eglitis, et al. (1985) Science 230:1395-1398;Danos and Mulligan (1988) Proc. Natl. Acad. Sci. USA 85:6460-6464;Wilson et al. (1988) Proc. Natl. Acad. Sci. USA 85:3014-3018; Armentanoet al. (1990) Proc. Natl. Acad. Sci. USA 87:6141-6145; Huber et al.(1991) Proc. Natl. Acad. Sci. USA 88:8039-8043; Ferry et al. (1991)Proc. Natl. Acad. Sci. USA 88:8377-8381; Chowdhury et al. (1991) Science254:1802-1805; van Beusechem et al. (1992) Proc. Natl. Acad. Sci. USA89:7640-7644; Kay et al. (1992) Human Gene Therapy 3:641-647; Dai et al.(1992) Proc. Natl. Acad. Sci. USA 89:10892-10895; Hwu et al. (1993) J.Immunol. 150:4104-4115; U.S. Pat. No. 4,868,116; U.S. Pat. No.4,980,286; PCT Application WO 89/07136; PCT Application WO 89/02468; PCTApplication WO 89/05345; and PCT Application WO 92/07573). Retroviralvectors require target cell division in order for the retroviral genome(and foreign nucleic acid inserted into it) to be integrated into thehost genome to stably introduce nucleic acid into the cell. Thus, it maybe necessary to stimulate replication of the target cell.

Adenoviruses:

The genome of an adenovirus can be manipulated such that it encodes andexpresses a gene product of interest but is inactivated in terms of itsability to replicate in a normal lytic viral life cycle. See for exampleBerkner et al (1988) BioTechniques 6:616; Rosenfeld et al. (1991)Science 252:431-434; and Rosenfeld et al. (1992) Cell 68:143-155.Suitable adenoviral vectors derived from the adenovirus strain Ad type 5dl324 or other strains of adenovirus (e.g., Ad2, Ad3, Ad7 etc.) are wellknown to those skilled in the art. Recombinant adenoviruses areadvantageous in that they do not require dividing cells to be effectivegene delivery vehicles and can be used to infect a wide variety of celltypes, including airway epithelium (Rosenfeld et al (1992) cited supra),endothelial cells (Lemarchand et al. (1992) Proc. Natl. Acad. Sci. USA89:6482-6486), hepatocytes (Herz and Gerard (1993) Proc. Natl. Acad.Sci. USA 90:2812-2816) and muscle cells (Quantin et al. (1992) Proc.Natl. Acad. Sci. USA 89:2581-2584). Additionally, introduced adenoviralDNA (and foreign DNA contained therein) is not integrated into thegenome of a host cell but remains episomal, thereby avoiding potentialproblems that can occur as a result of insertional mutagenesis insituations where introduced DNA becomes integrated into the host genome(e.g., retroviral DNA). Moreover, the carrying capacity of theadenoviral genome for foreign DNA is large (up to 8 kilobases) relativeto other gene delivery vectors (Berkner et al cited supra; Haj-Ahmandand Graham (1986) J. Virol. 57:267). Most replication-defectiveadenoviral vectors currently in use are deleted for all or parts of theviral E1 and E3 genes but retain as much as 80% of the adenoviralgenetic material.

Adeno-associated Viruses:

Adeno-associated virus (AAV) is a naturally occurring defective virusthat requires another virus, such as an adenovirus or a herpes virus, asa helper virus for efficient replication and a productive life cycle.(For a review see Muzyczka et al Curr. Topics in Micro. and Immunol.(1992) 158:97-129). It is also one of the few viruses that may integrateits DNA into non-dividing cells, and exhibits a high frequency of stableintegration (see for example Flotte et al. (1992) Am. J. Respir. Cell.Mol. Biol. 7:349-356; Samulski et al. (1989) J. Virol. 63:3822-3828; andMcLaughlin et al. (1989) J. Virol. 62:1963-1973). Vectors containing aslittle as 300 base pairs of AAV can be packaged and can integrate. Spacefor exogenous DNA is limited to about 4.5 kb. An AAV vector such as thatdescribed in Tratschin et al. (1985) Mol. Cell. Biol. 5:3251-3260 can beused to introduce DNA into cells. A variety of nucleic acids have beenintroduced into different cell types using AAV vectors (see for exampleHermonat et al. (1984) Proc. Natl. Acad. Sci. USA 81:6466-6470;Tratschin et al. (1985) Mol. Cell. Biol. 4:2072-2081; Wondisford et al.(1988) Mol. Endocrinol. 2:32-39; Tratschin et al. (1984) J. Virol.51:611-619; and Flotte et al. (1993) J. Biol. Chem. 268:3781-3790).

The efficacy of a particular expression vector system and method ofintroducing nucleic acid into a cell can be assessed by standardapproaches routinely used in the art. For example, DNA introduced into acell can be detected by a filter hybridization technique (e.g., Southernblotting) and RNA produced by transcription of introduced DNA can bedetected, for example, by Northern blotting, RNase protection or reversetranscriptase-polymerase chain reaction (RT-PCR). The gene product canbe detected by an appropriate assay, for example by immunologicaldetection of a produced protein, such as with a specific antibody, or bya functional assay to detect a functional activity of the gene product.

In a preferred embodiment, a retroviral expression vector encoding humanc-Maf is used to express human c-Maf protein in cells in vivo, tothereby stimulate c-Maf protein activity in vivo. Such retroviralvectors can be prepared according to standard methods known in the art(discussed further above).

A modulatory agent, such as a chemical compound, can be administered toa subject as a pharmaceutical composition. Such compositions typicallycomprise the modulatory agent and a pharmaceutically acceptable carrier.As used herein the term “pharmaceutically acceptable carrier” isintended to include any and all solvents, dispersion media, coatings,antibacterial and antifungal agents, isotonic and absorption delayingagents, and the like, compatible with pharmaceutical administration. Theuse of such media and agents for pharmaceutically active substances iswell known in the art. Except insofar as any conventional media or agentis incompatible with the active compound, use thereof in thecompositions is contemplated. Supplementary active compounds can also beincorporated into the compositions. Pharmaceutical compositions can beprepared as described above in subsection IV.

The identification of c-Maf as a key regulator of the production of IL-4(see e.g., PCT Publication WO 97/39721 and Ho, I-C. et al. (1996) Cell85:973-983), and hence continued formation of Th2 cells, allows forselective manipulation of T cell subsets in a variety of clinicalsituations using the modulatory methods of the invention. Thestimulatory methods of the invention (i.e., methods that use astimulatory agent to enhance human c-Maf activity) result in productionof Th2-associated cytokines, with concomitant promotion of a Th2response and downregulation of a Th1 response. In contrast, theinhibitory methods of the invention (i.e., methods that use aninhibitory agent to downmodulate human c-Maf activity) inhibit theproduction of Th2-associated cytokines, with concomitant downregulationof a Th2 response and promotion of a Th1 response. Thus, to treat adisease condition wherein a Th2 response is beneficial, a stimulatorymethod of the invention is selected such that Th2 responses are promotedwhile downregulating Th1 responses. Alternatively, to treat a diseasecondition wherein a Th1 response is beneficial, an inhibitory method ofthe invention is selected such that Th2 responses are downregulatedwhile promoting Th1 responses. Application of the methods of theinvention to the treatment of disease conditions may result in cure ofthe condition, a decrease in the type or number of symptoms associatedwith the condition, either in the long term or short term (i.e.,amelioration of the condition) or simply a transient beneficial effectto the subject.

Numerous disease conditions associated with a predominant Th1 orTh2-type response have been identified and could benefit from modulationof the type of response mounted in the individual suffering from thedisease condition. Application of the immunomodulatory methods of theinvention to such diseases is described in further detail below.

A. Allergies

Allergies are mediated through IgE antibodies whose production isregulated by the activity of Th2 cells and the cytokines producedthereby. In allergic reactions, IL-4 is produced by Th2 cells, whichfurther stimulates production of IgE antibodies and activation of cellsthat mediate allergic reactions, i.e., mast cells and basophils. IL-4also plays an important role in eosinophil mediated inflammatoryreactions. Accordingly, the inhibitory methods of the invention can beused to inhibit the production of Th2-associated cytokines, and inparticular IL-4, in allergic patients as a means to downregulateproduction of pathogenic IgE antibodies. An inhibitory agent may bedirectly administered to the subject or cells (e.g., Thp cells or Th2cells) may be obtained from the subject, contacted with an inhibitoryagent ex vivo, and readministered to the subject. Moreover, in certainsituations it may be beneficial to coadminister to the subject theallergen together with the inhibitory agent or cells treated with theinhibitory agent to inhibit (e.g. desensitize) the allergen-specificresponse. The treatment may be further enhanced by administering otherTh1-promoting agents, such as the cytokine IL-12 or antibodies toTh2-associated cytokines (e.g., anti-IL-4 antibodies), to the allergicsubject in amounts sufficient to further stimulate a Th1-type response.

B. Cancer

The expression of Th2-promoting cytokines has been reported to beelevated in cancer patients (see e.g., Yamamura, M., et al. (1993) J.Clin. Invest. 91:1005-1010; Pisa, P., et al. (1992) Proc. Natl. Acad.Sci. USA 89:7708-7712) and malignant disease is often associated with ashift from Th1 type responses to Th2 type responses along with aworsening of the course of the disease. Accordingly, the inhibitorymethods of the invention can be used to inhibit the production ofTh2-associated cytokines in cancer patients, as a means to counteractthe Th1 to Th2 shift and thereby promote an ongoing Th1 response in thepatients to ameliorate the course of the disease. The inhibitory methodcan involve either direct administration of an inhibitory agent to asubject with cancer or ex vivo treatment of cells obtained from thesubject (e.g., Thp or Th2 cells) with an inhibitory agent followed byreadministration of the cells to the subject. The treatment may befurther enhanced by administering other Th1-promoting agents, such asthe cytokine IL-12 or antibodies to Th2-associated cytokines (e.g.,anti-IL-4 antibodies), to the recipient in amounts sufficient to furtherstimulate a Th1-type response.

C. Infectious Diseases

The expression of Th2-promoting cytokines also has been reported toincrease during a variety of infectious diseases, including HIVinfection, tuberculosis, leishmaniasis, schistosomiasis, filarialnematode infection and intestinal nematode infection (see e.g.; Shearer,G. M. and Clerici, M. (1992) Prog. Chem. Immunol. 54:21-43; Clerici, Mand Shearer, G. M. (1993) Immunology Today 14:107-111; Fauci, A. S.(1988) Science 239:617-623; Locksley, R. M. and Scott, P. (1992)Immunoparasitology Today 1:A58-A61; Pearce, E. J., et al. (1991) J. Exp.Med. 173:159-166; Grzych, J-M., et al. (1991) J. Immunol. 141:1322-1327;Kullberg, M. C., et al. (1992) J. Immunol. 148:3264-3270; Bancroft, A.J., et al. (1993) J. Immunol. 150: 1395-1402; Pearlman, E., et al.(1993) Infect. Immun. 61:1105-1112; Else, K. J., et al. (1994) J. Exp.Med. 179:347-351) and such infectious diseases are also associated witha Th1 to Th2 shift in the immune response. Accordingly, the inhibitorymethods of the invention can be used to inhibit the production ofTh2-associated cytokines in subjects with infectious diseases, as ameans to counteract the Th1 to Th2 shift and thereby promote an ongoingTh1 response in the patients to ameliorate the course of the infection.The inhibitory method can involve either direct administration of aninhibitory agent to a subject with an infectious disease or ex vivotreatment of cells obtained from the subject (e.g., Thp or Th2 cells)with an inhibitory agent followed by readministration of the cells tothe subject. The treatment may be further enhanced by administeringother Th1-promoting agents, such as the cytokine IL-12 or antibodies toTh2-associated cytokines (e.g., anti-IL-4 antibodies), to the recipientin amounts sufficient to further stimulate a Th1-type response.

D. Autoimmune Diseases

The stimulatory methods of the invention can be used therapeutically inthe treatment of autoimmune diseases that are associated with a Th2-typedysfunction. Many autoimmune disorders are the result of inappropriateactivation of T cells that are reactive against self tissue and thatpromote the production of cytokines and autoantibodies involved in thepathology of the diseases. Modulation of T helper-type responses canhave an effect on the course of the autoimmune disease. For example, inexperimental allergic encephalomyelitis (EAE), stimulation of a Th2-typeresponse by administration of IL-4 at the time of the induction of thedisease diminishes the intensity of the autoimmune disease (Paul, W. E.,et al. (1994) Cell 76:241-251). Furthermore, recovery of the animalsfrom the disease has been shown to be associated with an increase in aTh2-type response as evidenced by an increase of Th2-specific cytokines(Koury, S. J., et al. (1992) J. Exp. Med. 176:1355-1364). Moreover, Tcells that can suppress EAE secrete Th2-specific cytokines (Chen, C., etal. (1994) Immunity 1:147-154). Since stimulation of a Th2-type responsein EAE has a protective effect against the disease, stimulation of a Th2response in subjects with multiple sclerosis (for which EAE is a model)is likely to be beneficial therapeutically.

Similarly, stimulation of a Th2-type response in type I diabetes in miceprovides a protective effect against the disease. Indeed, treatment ofNOD mice with IL-4 (which promotes a Th2 response) prevents or delaysonset of type I diabetes that normally develops in these mice (Rapoport,M. J., et al. (1993) J. Exp. Med. 178:87-99). Thus, stimulation of a Th2response in a subject suffering from or susceptible to diabetes mayameliorate the effects of the disease or inhibit the onset of thedisease.

Yet another autoimmune disease in which stimulation of a Th2-typeresponse may be beneficial is rheumatoid arthritis (RA). Studies haveshown that patients with rheumatoid arthritis have predominantly Th1cells in synovial tissue (Simon, A. K., et al., (1994) Proc. Natl. Acad.Sci. USA 91:8562-8566). By stimulating a Th2 response in a subject withRA, the detrimental Th1 response can be concomitantly downmodulated tothereby ameliorate the effects of the disease.

Accordingly, the stimulatory methods of the invention can be used tostimulate production of Th2-associated cytokines in subjects sufferingfrom, or susceptible to, an autoimmune disease in which a Th2-typeresponse is beneficial to the course of the disease. The stimulatorymethod can involve either direct administration of a stimulatory agentto the subject or ex vivo treatment of cells obtained from the subject(e.g., Thp, Th1 cells, B cells, non-lymphoid cells) with a stimulatoryagent followed by readministration of the cells to the subject. Thetreatment may be further enhanced by administering other Th2-promotingagents, such as IL-4 itself or antibodies to Th1-associated cytokines,to the subject in amounts sufficient to further stimulate a Th2-typeresponse.

In contrast to the autoimmune diseases described above in which a Th2response is desirable, other autoimmune diseases may be ameliorated by aTh1-type response. Such diseases can be treated using an inhibitoryagent of the invention (as described above for cancer and infectiousdiseases). The treatment may be further enhanced by administrating aTh1-promoting cytokine (e.g., IFN-γ) to the subject in amountssufficient to further stimulate a Th 1-type response.

The efficacy of agents for treating autoimmune diseases can be tested inthe above described animal models of human diseases (e.g., EAE as amodel of multiple sclerosis and the NOD mice as a model for diabetes) orother well characterized animal models of human autoimmune diseases.Such animal models include the mrl/lpr/lpr mouse as a model for lupuserythematosus, murine collagen-induced arthritis as a model forrheumatoid arthritis, and murine experimental myasthenia gravis (seePaul ed., Fundamental Immunology, Raven Press, New York, 1989, pp.840-856). A modulatory (i.e., stimulatory or inhibitory) agent of theinvention is administered to test animals and the course of the diseasein the test animals is then monitored by the standard methods for theparticular model being used. Effectiveness of the modulatory agent isevidenced by amelioration of the disease condition in animals treatedwith the agent as compared to untreated animals (or animals treated witha control agent).

Non-limiting examples of autoimmune diseases and disorders having anautoimmune component that may be treated according to the inventioninclude diabetes mellitus, arthritis (including rheumatoid arthritis,juvenile rheumatoid arthritis, osteoarthritis, psoriatic arthritis),multiple sclerosis, myasthenia gravis, systemic lupus erythematosis,autoimmune thyroiditis, dermatitis (including atopic dermatitis andeczematous dermatitis), psoriasis, Sjögren's Syndrome, includingkeratoconjunctivitis sicca secondary to Sjögren's Syndrome, alopeciaareata, allergic responses due to arthropod bite reactions, Crohn'sdisease, aphthous ulcer, iritis, conjunctivitis, keratoconjunctivitis,ulcerative colitis, asthma, allergic asthma, cutaneous lupuserythematosus, scleroderma, vaginitis, proctitis, drug eruptions,leprosy reversal reactions, erythema nodosum leprosum, autoimmuneuveitis, allergic encephalomyelitis, acute necrotizing hemorrhagicencephalopathy, idiopathic bilateral progressive sensorineural hearingloss, aplastic anemia, pure red cell anemia, idiopathicthrombocytopenia, polychondritis, Wegener's granulomatosis, chronicactive hepatitis, Stevens-Johnson syndrome, idiopathic sprue, lichenplanus, Crohn's disease, Graves ophthalmopathy, sarcoidosis, primarybiliary cirrhosis, uveitis posterior, and interstitial lung fibrosis.

E. Transplantation

While graft rejection or graft acceptance may not be attributableexclusively to the action of a particular T cell subset (i.e., Th1 orTh2 cells) in the graft recipient (for a discussion see Dallman, M. J.(1995) Curr. Opin. Immunol. 7:632-638), numerous studies have implicateda predominant Th2 response in prolonged graft survival or a predominantTh2 response in graft rejection. For example, graft acceptance has beenassociated with production of a Th2 cytokine pattern and/or graftrejection has been associated with production of a Th1 cytokine pattern(see e.g., Takeuchi, T. et al. (1992) Transplantation 53:1281-1291;Tzakis, A. G. et al. (1994) J. Pediatr. Surg. 29:754-756; Thai, N. L. etal. (1995) Transplantation 59:274-281). Additionally, adoptive transferof cells having a Th2 cytokine phenotype prolongs skin graft survival(Maeda, H. et al. (1994) Int. Immunol. 6:855-862) and reducesgraft-versus-host disease (Fowler, D. H. et al. (1994) Blood84:3540-3549; Fowler, D. H. et al. (1994) Prog. Clin. Biol. Res.389:533-540). Still further, administration of IL-4, which promotes Th2differentiation, prolongs cardiac allograft survival (Levy, A. E. andAlexander, J. W. (1995) Transplantation 60:405-406), whereasadministration of IL-12 in combination with anti-IL-10 antibodies, whichpromotes Th1 differentiation, enhances skin allograft rejection(Gorczynski, R. M. et al. (1995) Transplantation 60:1337-1341).

Accordingly, the stimulatory methods of the invention can be used tostimulate production of Th2-associated cytokines in transplantrecipients to prolong survival of the graft. The stimulatory methods canbe used both in solid organ transplantation and in bone marrowtransplantation (e.g., to inhibit graft-versus-host disease). Thestimulatory method can involve either direct administration of astimulatory agent to the transplant recipient or ex vivo treatment ofcells obtained from the subject (e.g., Thp, Th1 cells, B cells,non-lymphoid cells) with a stimulatory agent followed byreadministration of the cells to the subject. The treatment may befurther enhanced by administering other Th2-promoting agents, such as

IL-4 itself or antibodies to Th1-associated cytokines, to the recipientin amounts sufficient to further stimulate a Th2-type response.

In addition to the foregoing disease situations, the modulatory methodsof the invention also are useful for other purposes. For example, thestimulatory methods of the invention (i.e., methods using a stimulatoryagent) can be used to stimulate production of Th2-promoting cytokines(e.g., IL-4) in vitro for commercial production of these cytokines(e.g., cells can be contacted with the stimulatory agent in vitro tostimulate IL-4 production and the IL-4 can be recovered from the culturesupernatant, further purified if necessary, and packaged for commercialuse).

Furthermore, the modulatory methods of the invention can be applied tovaccinations to promote either a Th1 or a Th2 response to an antigen ofinterest in a subject. That is, the agents of the invention can serve asadjuvants to direct an immune response to a vaccine either to a Th1response or a Th2 response. For example, to stimulate an antibodyresponse to an antigen of interest (i.e., for vaccination purposes), theantigen and a stimulatory agent of the invention can be coadministeredto a subject to promote a Th2 response to the antigen in the subject,since Th2 responses provide efficient B cell help and promote IgG1production. Alternatively, to promote a cellular immune response to anantigen of interest, the antigen and an inhibitory agent of theinvention can be coadministered to a subject to promote a Th1 responseto the antigen in a subject, since Th1 responses favor the developmentof cell-mediated immune responses (e.g., delayed hypersensitivityresponses). The antigen of interest and the modulatory agent can beformulated together into a single pharmaceutical composition or inseparate compositions. In a preferred embodiment, the antigen ofinterest and the modulatory agent are administered simultaneously to thesubject. Alternatively, in certain situations it may be desirable toadminister the antigen first and then the modulatory agent or vice versa(for example, in the case of an antigen that naturally evokes a Th1response, it may be beneficial to first administer the antigen alone tostimulate a Th1 response and then administer a stimulatory agent, aloneor together with a boost of antigen, to shift the immune response to aTh2 response).

This invention is further illustrated by the following example, whichshould not be construed as limiting. The contents of all references,patents and published patent applications cited throughout thisapplication are hereby incorporated by reference. Additionally, allnucleotide and amino acid sequences deposited in public databasesreferred to herein are also hereby incorporated by reference.

EXAMPLE Isolation and Characterization of a Human c-Maf Nucleic Acid

To isolate a nucleic acid molecule encoding human c-maf, a human genomicDNA library in lambda phage (commercially available from Stratagene) wasscreened with a radiolabeled DNA probe derived from the 3′ untranslatedregion of the mouse c-Maf gene. Following hybridization under standardconditions, filters were washed under stringent conditions in 0.2×SSC,0.1% SDS wash buffer at approximately 62° C. Phage clones that remainedhybridized to the probe under these conditions were selected andisolated to purity. The genomic inserts of the isolated phage weresubcloned into the plasmid vector pBluescript KS/II, by restrictiondigestion of the phage DNA with NheI and insertion into the XbaI site ofthe plasmid. E. coli bacterial cells carrying a pBluescript plasmidcontaining the human c-Maf coding region, referred to herein aspHu-c-Maf, has been deposited under the provisions of the BudapestTreaty with the American Type Culture Collection, Rockville, Md., onFeb. 24, 1998 and assigned ATCC Accession No. 98671. This plasmidcontains an NheI fragment of approximately 4.2 kb (derived from theisolated phage), cloned into the compatible XbaI site of the plasmidvector, to thereby create a ˜4.2 kb NheI/XbaI insert that encodes humanc-Maf. It should be noted that upon ligation of the NheI fragment intothe XbaI site, these restriction sites are not regenerated and, thus, toexcise the fragment from the plasmid, it is necessary to use adjacentrestriction sites within the pBluescript polylinker.

The coding region of human c-Maf, contained in the pHu-c-Maf plasmid,was sequenced by standard dideoxy sequencing methods. The nucleotide andpredicted amino acid sequences are shown in SEQ ID NOs: 1 and 2,respectively.

The coding region encompasses approximately 1.2 kb of DNA and thus, theremainder of the 4.2 kb insert of pHu-c-Maf represents 5′ and 3′untranslated sequences. FIG. 1 shows a comparison of the nucleotidesequence of hu-c-Maf shown of SEQ ID NO: 1 with the mouse c-Maf codingregion. A number of nucleotide differences between the two codingregions are evident, which differences are boxed in FIG. 1. FIG. 2 showsa comparison of the amino acid sequence of hu-c-Maf shown of SEQ ID NO:2 with the mouse c-Maf amino acid sequence. Again, a number of aminoacid differences between the two proteins are evident, which differencesare boxed in FIG. 2. The overall structure of the human c-Maf protein,however, is conserved with the mouse c-Maf protein, including thepresence of a leucine zipper domain at amino acid positions 313-348 ofSEQ ID NO: 2.

Equivalents

Those skilled in the art will recognize, or be able to ascertain usingno more than routine experimentation, many equivalents to the specificembodiments of the invention described herein. Such equivalents areintended to be encompassed by the following claims.

1. A method for identifying a compound that modulates the activity of ahuman c-Maf protein, comprising providing an indicator cell thatcomprises a recombinant expression vector encoding a human c-Maf proteinof SEQ ID NO.:2, wherein the human c-Maf-coding sequences areoperatively linked to regulatory sequences that allow for constitutiveexpression of human c-Maf in the indicator cell, and a reporter generesponsive to the human c-Maf protein; contacting the indicatorcomposition with a test compound; and determining the effect of the testcompound on the activity of the human c-Maf protein in the indicatorcell wherein the step of determining comprises evaluating the expressionof the reporter gene in the presence and absence of the test compound,to thereby identify a compound that modulates the activity of a humanc-Maf protein.
 2. The method of claim 1, wherein the reporter genecomprises nucleotides −157 to +58 relative to the +1 start site oftranscription of the interleukin-4 gene.
 3. The method of claim 1,wherein the reporter gene comprises about 3 kb of upstream regulatorysequences of the interleukin-4 gene.
 4. The method of claim 1, whereinthe reporter gene is selected from the group consisting of genes thatencode: chloramphenicol acetyltransferase, beta-galactosidase, alkalinephosphatase and luciferase.
 5. The method of claim 1, wherein theindicator cell does not normally express human c-Maf.
 6. The method ofclaim 1, wherein the indicator cell is a B cell.
 7. The method of claim6, wherein the indicator cell is a M12 B lymphoma cell.
 8. The method ofclaim 1, wherein the indicator cell is a Th1 cell clone.
 9. The methodof claim 8, wherein the indicator cell is an AE7 cells.
 10. The methodof claim 1, wherein the indicator cell is a nonlymphoid cell.
 11. Themethod of claim 10, wherein the indicator cell is a HEPG2 hepatoma cell.12. The method of claim 10, wherein the indicator cell is a yeast cell.13. A method for identifying a compound that modulates an immuneresponse, comprising providing an indicator cell that comprises arecombinant expression vector encoding a human c-Maf protein of SEQ IDNO.:2, wherein the human c-Maf-coding sequences are operatively linkedto regulatory sequences that allow for constitutive expression of humanc-Maf in the indicator cell, and a Th2-associated cytokine generesponsive to the human c-Maf protein; contacting the indicatorcomposition with a test compound; and determining the effect of the testcompound on an immune response, wherein the step of determiningcomprises evaluating the effect of the compound on expression of theTh2-associated cytokine gene in the presence and the absence of the testcompound, to thereby identify a compound that modulates an immuneresponse.
 14. The method of claim 13, wherein the effect of the testcompound on an immune response is determined by determining the effectof the compound on production of a Th2-associated cytokine protein. 15.The method of claim 14, wherein the Th2-associated cytokine gene is aninterleukin-4 gene.
 16. The method of claim 13, wherein the effect ofthe test compound on the expression of a Th2-associated cytokine gene isdetermined by determining the effect of the compound on the developmentof T helper type 1 (Th1) cells.
 17. The method of claim 13, wherein theeffect of the test compound on the expression of a Th2-associatedcytokine gene is determined by determining the effect of the compound onthe development of T helper type 2 (Th2) cells.
 18. The method of claim1, wherein the reporter gene is a Th2-associated cytokine.
 19. Themethod of claim 13 or 18, wherein the Th2-associated cytokine isinterleukin-4.
 20. The method of claim 1 or 13, wherein the reportergene is operatively linked to regulatory sequences of a Th2-associatedcytokine gene.
 21. The method of claim 1 or 13, wherein the human c-Mafcoding sequences are operatively linked to regulatory sequences of theendogenous human c-Maf gene, wherein the regulatory sequences of theendogenous human c-Maf gene comprise the untranslated sequences of theNheI/XbaI fragment of pHu-c-Maf (ATCC Accession No. 98671).
 22. A methodfor identifying a compound that modulates the activity of a human c-Mafprotein comprising, providing an indicator cell comprising a recombinantexpression vector encoding a human c-Maf protein comprising theNheI/XbaI fragment of pHu-c-Maf (ATCC Accession No. 98671), wherein thehuman c-Maf-coding sequences are operatively linked to regulatorysequences that allow for constitutive expression of human c-Maf in theindicator cell, and a cytokine gene responsive to the human c-Mafprotein, contacting the indicator cell with a test compound, anddetermining the effect of the test compound on human c-Maf activity byevaluating the level of cytokine production in the indicator cell in thepresence and absence of the test compound, wherein a modulation of thelevel of cytokine production identifies the test compound as a modulatorof the activity of a human c-Maf protein.
 23. The method of claim 22,wherein the level of cytokine production is determined by detectingcytokine mRNA in the indicator cell.
 24. The method of claim 22, whereinthe level of cytokine production is determined by detecting cytokinesecretion into the culture supernatant.
 25. The method of claim 22,wherein the cytokine is interleukin-4.